Pharmaceutical compositions and methods to vaccinate against disseminated candidiasis and other infectious agents

ABSTRACT

The invention provides a vaccine including an isolated Als protein family member having cell adhesion activity, or an immunogenic fragment thereof, with an adjuvant in a pharmaceutically acceptable medium. The invention also provides a method of treating or preventing hematogenously disseminated or mucocutaneous candidiasis. The method includes administering an immunogenic amount of a vaccine an isolated Als protein family member having cell adhesion activity, or an immunogenic fragment thereof, in a pharmaceutically acceptable medium. A method of treating or preventing disseminated candidiasis also is provided that includes administering an effective amount of an isolated Als protein family member having cell adhesion activity, or an functional fragment thereof, to inhibit the binding or invasion of  Candida  to a host cell or tissue. The Als protein family member can be derived from a  Candida  strain selected from the group consisting of  Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata  and  Candida parapsilosis  and the Als protein family member includes Als1p, Als3p, Als5p, Als6p, Als7p or Als9p. Also provided is a method of treating or preventing  Staphylococcus aureus  infections. The method includes administering an immunogenic amount of a vaccine an isolated Als protein family member having cell adhesion activity, or an immunogenic fragment thereof, in a pharmaceutically acceptable medium.

This application is a continuation-in-part application of U.S. Ser. No.11/123,875, filed May 5, 2005, which is a continuation-in-partapplication of U.S. Ser. No. 10/245,802, filed Sep. 13, 2002, which is acontinuation-in-part of U.S. Ser. No. 09/715,876, filed Nov. 18, 2000,which is based on, and claims the benefit of, U.S. ProvisionalApplication Ser. No. 60/166,663 filed Nov. 19, 1999, all of which areherein incorporated by reference in its entirety.

This invention was made with government support under Public HealthService grants PO-1AI-37194, RO1Ai-19990, and MO1 RR0425. The UnitedStates Government has certain rights in this invention

BACKGROUND OF THE INVENTION

This invention relates to Candida albicans surface adhesin proteins, toantibodies resulting from an immune response to vaccination with C.albicans surface adhesion proteins and to methods for the preventionand/or treatment of candidiasis and other bacterial infections with C.albicans surface adhesion proteins.

There has been a dramatic increase in the incidence of nosocomialinfections caused by Candida species in recent years. The incidence ofhematogenously disseminated candidal infections increased 11-fold from1980 to 1989. This increasing incidence has continued into the 1990s.Infections by Candida species are now the fourth most common cause ofnosocomial septicemia, are equal to that of Escherichia coli, andsurpass the incidence caused by Klebsiella species. Furthermore Candidaspecies are the most common cause of deep-seated fungal infections inpatients who have extensive burns. Up to 11% of individuals undergoingbone marrow transplantation and 13% of those having an orthotopic livertransplant will develop an invasive candidal infection.

Candida albicans, the major pathogen in this genus, can switch betweentwo morphologies: the blastospore (budding yeast) and filamentous(hyphae and pseudohyphae) phases. Candida mutants that are defective ingenes regulating filamentation are reported to have reduced virulence inanimal models. This reduced virulence suggests that the ability tochange from a blastospore to a filament is a key virulence factor of C.albicans. To date, no essential effectors of these filamentationpathways have been identified in C. albicans. See Caesar-TonThat, T. C.and J. E. Cutler, “A monoclonal antibody to Candida albicans enhancesmouse neutrophil candidacidal activity,” Infect. Immun. 65:5354-5357,1997.

Staphylococcus aureus infections also are common and increasingly resultin drug resistance to antibiotics. For example, S. aureus is a commoncause of skin and skin structure infections, endocarditis and bacteremiain the U.S. and throughout the world. Formerly community acquired S.aureus (CA-S. aureus) infections were nearly uniformly susceptible topenicillinase-resistant beta lactams such as cefazolin, oxacillin,methicillin, penicillin and amoxicillin. However, over the past decade,epidemics of beta-lactam resistant S. aureus (MRSA) infection have beenseen in multiple locales throughout the world, especially communityacquired MRSA (CA-MRSA). In many places MRSA has become the predominantS. aureus strain causing CA infections. A recent, prospective,population-based survey in three states in the U.S. estimated that theincidence of CA-MRSA infections is 500 cases per 100,000 population,which translates to approximately 1.5 million cases per year in the U.S.alone. The increasing frequency of drug-resistant S. aureus infectionshighlights the need for new ways to prevent and treat these infections.

The identification of effectors in the regulatory pathways of theorganism that contribute to virulence offers the opportunity fortherapeutic intervention with methods or compositions that are superiorto existing antifungal agents. The identification of cell surfaceproteins that affect a regulatory pathway involved in virulence isparticularly promising because characterization of the protein enableimmunotherapeutic techniques that are superior to existing antifungalagents when fighting a candidal infection.

The virulence of Candida albicans is regulated by several putativevirulence factors of which adherence to host constituents and theability to transform from yeast-to-hyphae are among the most critical indetermining pathogenicity. While potent antifungal agents exist that aremicrobicidal for Candida, the attributable mortality of candidemia isapproximately 38%, even with treatment with potent anti-fungal agentssuch as amphotericin B. Also, existing agents such as amphotericin Btend to exhibit undesirable toxicity. Although additional antifungalsmay be developed that are less toxic than amphotericin B, it is unlikelythat agents will be developed that are more potent. Therefore, eitherpassive or active immunotherapy to treat or prevent disseminatedcandidiasis is a promising alternative to standard antifungal therapy.

Thus, there exists a need for effective immunogens that will providehost immune protection and passive immunoprotection against Candida, S.aureus and other immunogenically related pathogens. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a vaccine including an isolated Als proteinfamily member having cell adhesion activity, or an immunogenic fragmentthereof, with an adjuvant in a pharmaceutically acceptable medium. Theinvention also provides a method of treating or preventing disseminatedcandidiasis. The method includes administering an immunogenic amount ofa vaccine an isolated Als protein family member having cell adhesionactivity, or an immunogenic fragment thereof, in a pharmaceuticallyacceptable medium. A method of treating or preventing disseminatedcandidiasis also is provided that includes administering an effectiveamount of an isolated Als protein family member having cell adhesionactivity, or an functional fragment thereof, to inhibit the binding orinvasion of Candida to a host cell or tissue. The Als protein familymember can be derived from a Candida strain selected from the groupconsisting of Candida albicans, Candida krusei, Candida tropicalis,Candida glabrata and Candida parapsilosis and the Als protein familymember includes Als1p, Als3p, Als5p, Als6p, Als7p or Als9p. Alsoprovided is a method of treating or preventing Staphylococcus aureusinfections. The method includes administering an immunogenic amount of avaccine an isolated Als protein family member having cell adhesionactivity, or an immunogenic fragment thereof, in a pharmaceuticallyacceptable medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, 1B show the mediation of Als1p adherence of C. albicans tohuman umbilical vein endothelial cells. Values represent the mean±SD ofat least three independent experiments, each performed in triplicate.(A) Endothelial cell adherence of ALS1l/als2 alsl/alsl andALS1-complemented mutants and wild-type CAI12(30)(B) Endothelial celladherence of P_(ADH1)-ALS1 mutant that overexpresses ALS1, compared towild type C. albicans. Statistical treatment was obtained by Wilcoxonran sum test and corrected for multiple comparisons with the Bonferronicorrection. *P<0.001 for all comparisons.

FIG. 2A-D shows the cell surface localization of Als1p on filaments ofC. albicans indirect immunofluorescence. Filamentation of C. albicanswas induced by incubating yeast cells in RPMI 1640 medium with glutaminefor 1.5 hours at 37° C. Als1p was detected by incubating organisms firstwith anti-Als1p mouse mAb followed by FITC-labeled goat anti-mouse IgG.C. albicans cell surface was also stained with anti-C. albicanspolyclonal Ab conjugated with Alexa 594 (Molecular Probes, Eugene,Oreg.). Areas with yellow staining represent Als1p localization. (A) C.albicans wild-type. (B) als1/als1 mutant strain. (C) als1/als1complemented with wild type ALS1 (D) P_(ADH1)-ALS1 overexpressionmutant.

FIG. 3A, 3B show the mediation of Als1p on C. albicans filamentation onsolid medium. C. albicans blastospores were spotted on Lee's agar platesand incubated at 37° C. for 4 days (A) or 3 days (B).

FIG. 4A, 4B show the control of ALSl expression and the mediation of C.albicans filamentation by the EFG1 filamentation regulatory pathway. (A)Northern blot analysis showing expression of ALS1 in (i) mutantsdeficient in different filamentation regulatory pathways. (ii) efg1/efg1mutant complemented with either EFG1 or P_(ADH1)-ALS1. Total RNA wasextracted from cells grown in RPM1 1640+glutaine medium at 37° C. for 90minutes to induce filamentation. Blots were probed with ALS1 and TEF1.(B) Photomicrographs of the efg1/efg1 mutant and efg1/efg1 mutantcomplemented with P_(ADH1)-ALS1 grown on Lee's agar plates at 37° C. for4 days.

FIG. 5A, 5B show the reduction of virulence in the mouse model ofhematogenously disseminated candidiasis by (A) Male Balb/C mice (n=30for each yeast strain) were injected with stationary phase blastospores(10⁶ per mouse in 0.5 ml of PBS). Curves are the compiled results ofthree replicate experiments (n=30 mice for each strain). The doublingtimes of all strains, grown in YPD at 30° C., ranged between 1.29 to1.52 hours and were not statistically different from each other.Southern blot analysis Of total chromosomal DNA was used to match theidentity of the genotype of C. albicans strains retrieved from infectedorgans with those of C. albicans strains used to infect the mice.Statistical analysis was obtained by Wilcoxon rank sum test andcorrected for multiple comparisons with the Bonferroni correction.*P<0.002 for the als1/als1 mutant versus each of the other strains. (B)Histological micrographs of kidneys infected with C. albicans wild-type,homozygous als1 null mutant, or heterozygous ALS1 complemented mutant.Kidney samples were retrieved 28 hours (a) or 40 (b) hours postinfection, fixed in paraformaldehyde and sections were stained withsilver (magnification ×400). Arrows denote C. albicans cells.

FIG. 6 shows the prophylactic effect of anti-ALS antibody againstdisseminated candidiasis as a function of surviving animals over a30-day period for animals infused with anti-Als1p polyserum.

FIG. 7 is polypeptide sequence alignment of the N-terminal portion ofselect ALS polypeptides arranged by adherence phenotype. The top threelines are the sequences from ALS1, 3 and 5 polypeptides (SEQ ID NOS:1-3, respectively), which bind endothelial cells. The bottom three aresequences from ALS6, 7 and 9 polypeptides (SEQ ID NOS; 4-6,respectively), which do not bind endothelial cells. The last linerepresents the ALS polypeptide family consensus sequence (SEQ ID NO:7).

FIG. 8 shows Als proteins confer substrate-specific adherence propertieswhen heterologously expressed in Saccharonzyces cerevisiae. Each paneldemonstrates the percentage adherence of one Alsp expression strain(filled bars) to a variety of substrates to which C. albicans is knownto adhere. Adherence of S. cerevisiae transformed with the empty vector(empty bars) is included in each panel as a negative control. Gel,gelatin; FN, fibronectin; LN, laminin; FaDU, FaDU epithelial cells; EC,endothelial cells. *, p<0.01 when compared with empty plasmid control bysingle factor analysis of variance. Results are the mean±S.D. of atleast three experiments performed in triplicate.

FIG. 9 shows domain swapping demonstrates that substrate-specificadherence is determined by the composition of the N-terminal domain ofAls proteins. A representation of the ALS gene or construct being testedis depicted as a bar composed of sequences from ALS5 (black) or ALS6(white). Adherence properties of each mutant are displayed as aphotomicrograph illustrating the adherence of transformed S. cerevisiaeto fibronectin-coated beads and a graph demonstrating the adherence togelatin (black bars) and endothelial cells (gray bars) as measured inthe 6-well plate assay. Results are mean±S.D. of at least threeexperiments, each performed in triplicate.

FIG. 10 shows a subset of Als proteins mediate endothelial cell invasionwhen expressed in S. cerevisiae. A, endothelial cell adherence of S.cerevisiae strains expressing Als proteins or transformed with the emptyplasmid (control). Data represent the total number of endothelialcell-associated organisms and are expressed as cells per high powerfield. B, degree of endothelial cell invasion of Alsp expressing S.cerevisiae strains presented as the number of intracellular organismsper high power field. *, p<0.01 when compared with empty plasmid controlby single factor analysis of variance. Results are the mean±S.D. of atleast three experiments performed in triplicate.

FIG. 11 shows an alignment of the N-terminal amino acid sequence of Alsproteins of known function demonstrates an alternating pattern of CRsand HVRs. A, percentage of consensus identity among the N-terminalregions of Als proteins of known function. Note that the signal peptideregion (amino acids 1-20) is not shown. Open boxes indicate the regionsdesignated as HVRs 1-7. B, schematic alignment of Als proteins (SEQ IDNOS:1-6, respectively) showing the composition of the individual HVRs.The sequences are arranged to compare proteins with an affinity tomultiple substrates with those that bind few or no identifiedsubstrates. The number of amino acids in each conserved region isindicated in parentheses.

FIG. 12 shows CD and FTIR spectra of the Als1 protein N-terminal domain.A, circular dichroism spectrum of 10 μM Als1p in phosphate-bufferedsaline. B, FTIR spectrum of Als1p self-film hydrated with D₂O vapor.

FIG. 13 shows a comparison of predicted physicochemical properties ofN-terminal domains among the Als protein family. Hydrophobic,electrostatic, or hydrogen-bonding features are projected ontowater-accessible surfaces of each domain. Hydrophobics are shown asfollows: brown, most hydrophobic; blue, most hydrophilic. Electrostatics(spectral continuum) is shown as follows: red, most positive charge (+10kcal/mol); blue, most negative charge (−10 kcal/mol). Hydrogen-bondingpotential (H-binding) is shOwn as follows: red, donor; blue, acceptor.Als proteins are distinguishable into three groups based on thecomposite of these properties. For example, note the similarhydrophobic, electrostatic, and hydrogen-bonding profiles among Alsgroup A proteins, Als1p, Als3p, and Als5p. In contrast, Als group Bmembers, Als6p and Als7p, display striking differences in hydrophobicand electrostatic features from those of Als group A. In addition tobiochemical profiles, note the differences in predicted structure amongthese domains.

FIG. 14. Conceptual model of structural-functional relationships in Alsfamily proteins. Als proteins are composed of three general components:an N-terminal domain, tandem repeats, and a serine/threonine-richC-terminal domain containing a glycosylphosphatidylinositol anchor thatinterfaces with the C. albicans cell wall. As illustrated, Als proteinscontain multiple conserved anti-parallel β-sheet regions (CR1-n) thatare interposed by extended spans, characteristic of the immunoglobulinsuperfamily. Projecting from the β-sheet domains are loop/coilstructures containing the HVRs. The three-dimensional physicochemicalproperties of specific Als protein HVRs probably govern interactionswith host substrates that confer adhesive and invasive functions toCandida. For illustrative purposes, only three N-terminal β-sheet/coildomains and their respective CR/HVR components are shown. Note that thisprojection is viewed at right angles to the structural images shown inFIG. 13.

FIG. 15. Immunization of mice (retired breeders) with rAls1p-N improvessurvival during subsequent disseminated candidiasis. Survival of miceimmunized with Als1p plus adjuvant. N=16 mice per group in duplicateexperiments on different days; Adj.=adjuvant. *p<0.05 vs adjuvant.

FIG. 16. Immunization with rAls1p-N improves the survival of bothretired breeder and juvenile mice. Survival of retired breeder (A) andjuvenile (B) mice infected with a rapidly fatal, 10⁶ inoculum of C.albicans. N=16 mice per group in duplicate experiments on differentdays; Adj.=adjuvant. *p<0.05 vs adjuvant control.

FIG. 17. Anti-rAls1p-N titers do not correlate with survival. Titers ofanti-rAls1p-N polyclonal antibodies raised in Balb/c mice immunized withvarying doses of rAls1p-N with or without adjuvant. Adj.=adjuvant. *p≦0.005 for 200 μg vs. all others.

FIG. 18. Only the protective dose of rAls1p-N induces an increase in C.albicans-stimulated Th1 splenocytes. Induction of Th1 (CD4⁺IFN-γ⁺IL-4⁻)and Th2 (CD4⁺IFN-γ⁻IL-4⁺) splenocytes by different doses of the rAls1p-Nvaccine. Splenocytes from immunized mice (n=9 per group) were stimulatedfor 48 h with heat-killed pre-germinated C. albicans and then analyzedby 3-color flow cytometry. *p=0.03 vs. adjuvant.

FIG. 19. Only the protective dose of rAls1p-N induces an increase inrAls1p-N-stimulated delayed type hypersensitivity. Delayed typehypersensitivity, assessed by footpad swelling, in mice (n=9-12 pergroup) vaccinated with rAls1p-N or CFA alone. Mice were immunized withthe indicated amount of rAls1p-N and then injected with 50 μg ofrAls1p-N into the footpad. Footpad swelling was assessed 24 h later.*p<0.05 versus adjuvant, 0.2 μg, and 200 μg.

FIG. 20. The rAls1p-N vaccine requires T cells, but not B cells, toinduce protective immunity. Survival of B cell-deficient, Tcell-deficient (nude), and congenic wild-type Balb/c control mice (n=7or 8per group) was simultaneously assessed after vaccination withrAls1p-N+adjuvant or adjuvant alone. *p<0.04 versus adjuvant alone,^(¶)p=0.003 versus wild-type adjuvant-treated.

FIG. 21. SQ vaccination with rAls1p-N induces an in vivo DTH response inimmunocompetent mice. Footpad swelling was assessed 24 h after injectionof 50 μg of rAls1p-N into the footpad in BALB/c mice (n=10 per group).Median values are displayed as black bars. *p=0.002 vs. control byWilcoxon Rank Sum test.

FIG. 22. The rAls1p-N vaccine improves survival of immunocompetent micewith hematogenously disseminated candidiasis and reduces tissue fungalburden. A) Survival of vaccinated or control BALB/c mice (n=7 or 10 pergroup for 2.5 or 5×10⁵ inocula, respectively) mice subsequently infectedvia the tail-vein with C. albicans. Each experiment was terminated at 30days post-infection with all remaining mice appearing well. *p<0.05 vs.Control by Log Rank test. B) Kidney fungal burden in BALB/c mice (n=7per group) infected via the tail vein with 5×10⁵ blastospores of C.albicans. The y axis reflects the lower limit of detection of the assay.Median values are displayed as black bars. *p=0.01 vs control byWilxocon Rank Sum test.

FIG. 23. The rAls1p-N vaccine induces a DTH reaction in neutropenic miceand improves their survival during subsequent hematogenouslydisseminated candidiasis. A) Footpad swelling was assessed 24 h afterinjection of 50 μg of rAls1p-N into the footpad in BALB/c mice (n=10 forControl, n=8 for rAls1p-N). * p=0.006 vs Control by Wilcoxon Rank Sumtest. B) Survival of neutropenic BALB/c mice (n=16 per group from 2experiments) infected with 2.5×10⁴ blastospores of C. albicans. *p=0.007vs adjuvant control by Log Rank test.

FIG. 24. The rAls1p-N vaccine diminishes the severity ofhistopathological fungal lesions on the tongues of mice withoropharyngeal candidiasis. N=4 mice per group. Inflammatory scoregenerated by a blinded observer as described in the text. *p=0.03 byWilcoxon Rank Sum test.

FIG. 25 shows that rAls3p-N but not rAls1p-N vaccine diminishes fungalcolonization of vagina of mice inoculated with C. albicans (*p=0.01 vsmice vaccinated with CFA alone, by Wilcoxon Rank Sum test) N=11 mice pergroup.

FIG. 26 shows an Als1p homology map versus S. aureus clumping factor A(c1n67A). Consensus functional sites from C. albicans Als1p and S.aureus ClfA were mapped onto the Als1p homology model. Numerous residuesfrom the N-termini of Als1p and ClfA map to a consensus cleft motif,which is where binding to substrate is predicted to occur for bothadhesins.

FIG. 27 shows that rAls1p-N and rAls3p-N vaccines improve the survivalof staphylococcemic mice. (*p<0.003 vs mice vaccinated with CFA alone,by Log Rank test). N 22 mice per group.

FIG. 28 shows that antibody titers do not correlate with degree ofprotection in individual vaccinated mice, but they do distinguishunvaccinated from vaccinated mice. Titers of anti-rAls1p-N oranti-rAls3p-N polyclonal antibodies raised in Balb/c mice immunized withCFA alone, or CFA+20 μg of rAls1p-N or rAls3p-N, respectively. Overallthere is a significant correlation between antibody titers and survival(rho=0.474, p=0.0057), indicating that antibody titers can be used as asurrogate marker for vaccine protection. However, when data from micereceiving CFA alone are excluded, there is no correlation betweenantibody titers and survival of mice vaccinated with rAls1p-N orrAls3p-N (rho 0.041143, p=0.847), indicating that antibodies are likelynot the predominant mechanism of protection of the vaccine.

FIG. 29 shows that the rAls1p-N vaccine protects outbred, CD1 mice fromhematogenously disseminated candidiasis. A) CD1 mice (n=8 per group)were vaccinated SQ with rAls1p-N (20 μg)+CFA, or CFA alone, and infectedvia the tail-vein with C. albicans SC5314 fourteen days after the boost.B) CD1 mice (n=8 per group) were vaccinated SQ with rAls1p-N at variousdoses with alum, or with alum alone, and infected via the tail-vein withC. albicans SC5314 fourteen days after the boost. * p<0.05 vs. adjuvantcontrol by Log Rank test.

FIG. 30 shows that the rAls1p-N vaccine improves the survival of Balb/cmice infected with one of several strains of C. albicans. Survival ofBalb/c mice immunized with rAls1p-N plus CFA versus CFA alone andinfected via the tail-vein with C. albicans 15563 (7×10⁵ blastospores),16240 (4×10⁵ blastospores), or 36082 (4×10⁵ blastospores) (n=8 mice pergroup). *p<0.05 ys adjuvant control by Log Rank test.

FIG. 31 shows that the rAls1p-N vaccine reduces tissue fungal burden inBalb/c mice infected with several non-albicans species of Candida.Balb/c mice (n=5 per group) were vaccinated with CFA or CFA+rAls1p-N (20μg) and infected via the tail-vein with C. glabrata, C. krusei, C.parapsilosis, or C. tropicalis. Infectious inocula are shown inparentheses below the species names. Kidney fungal burden was determinedon day five post-infection. The y axis reflects the lower limit ofdetection of the assay. *p<0.05 vs. adjuvant control by non-parametricSteel test for multiple comparisons.

FIG. 32 shows that rAls3p-N-immunized mice generated antibodies thatcross-reacted against rAls1p-N. Titers of individual mice immunized withCFA alone, CFA+rAls1p-N, or CFA+rAls3p-N. N=7 mice per group for CFA andCFA+rAls3p-N; n=6 mice for CFA+rAls1p-N. *p<0.05 vs. CFA alone;**p<0.002 vs. CFA alone & p<0.011 vs. CFA+rAls1p-N by Maim Whitney Utest. Bars denote medians.

FIG. 33 shows that both rAls1p-N and rAls3p-N primed mice for in vivodelayed type hypersensitivity responses. Mice (n=7 per group for CFA andCFA+rAls3p-N; n=6 for CFA+rAls1p-N) were vaccinated with CFA alone,CFA+rAls1p-N, or CFA+rAls3p-N. Delayed type hypersensitivity in vivo wasmeasured by footpad swelling. *p<0.05 vs. CFA alone by Mann Whitney Utest. Bars denote medians.

FIG. 34 shows that the rAls1p-N and rAls3p-N vaccines mediated similarefficacy against murine hematogenously disseminated candidiasis.Survival of Balb/c mice (n=15 per group from 2 experiments for CFA andCFA+rAls3p-N, and n=14 from 2 experiments for CFA+rAls1p-N) infected viathe tail vein with 5×10⁵ blastospores of C. albicans. The experiment wasterminated at day 28 post-infection with all remaining mice appearingwell. *p≦0.0001 vs CFA control by Log Rank test.

FIG. 35 shows that in vivo delayed-type hypersensitivity correlated withsurvival during disseminated candidiasis. Anti-rAls1p-N or anti-rAls3p-Nantibody titers and footpad swelling reactions were measured in mice(n=7 per group for CFA or CFA+rAls3p-N, n=6 for CFA+rAls1p-N) two daysprior to infection via the tail-vein with C. albicans. Correlationsdetermined with the Spearman Rank sum test.

FIG. 36 shows that the rAls3p-N vaccine significantly reduced tissuefungal burden during murine oropharyngeal candidiasis. Tongue fungalburden in mice (n=7 for CFA and 8 for rAls1p-N or rAls3p-N vaccinatedgroups) with oropharyngeal candidiasis. The y axis reflects the lowerlimit of detection of the assay. *p=0.005 vs. CFA by Mann Whitney Utest.

FIG. 37 shows that rAls3p-N reduced vaginal fungal burden compared toboth CFA alone and CFA+rAls1p-N in murine candidal vaginitis. Vaginalfungal burden in mice (n=11 per group from 2 experiments) vaccinatedwith CFA, CFA+rAls1p-N, or CFA+rAls3p-N. The y axis reflects the lowerlimit of detection of the assay. *p≦0.02 vs CFA and CFA+rAls1p-N bySteel test for multiple comparisons.

DETAILED DESCRIPTION OF THE INVENTION

Candida albicans and Staphylococcus aureus are common pathogen inhumans. For example, C. albicans, while normally a harmless commensal,this organism can cause a variety of conditions ranging from superficialmucocutaneous infection such as vaginal and/or oropharyngealcandidiasis, to deep organ involvement in disseminated candidiasis.Prior to causing disease, the fungus colonizes the gastrointestinaltract, and in some cases skin and mucous membranes. Adherence to hostmucosal surfaces is a key prerequisite for this initial step. Aftercolonization, C. albicans enters the bloodstream via infectedintravascular devices or by transmigration through gastrointestinalmucosa compromised by chemotherapy or stress ulcerations. Organisms thendisseminate via the bloodstream, bind to and penetrate the vascularendothelium to egress from the vascular tree, and invade deep organssuch as liver, spleen, and kidney.

The identification and functional characterizations of a variety ofexemplary Als protein family members described herein allow this familyof proteins to be effectively utilized in the treatment of candidiasis.Specific binding activity to diverse substrates and other selective celladhesion functions can be exploited in the production of vaccines foractive or passive immunization, in the production of peptide, analogueof mimetic inhibitors of cell adhesion to reduce or prevent initialinfection by inhibiting binding, adhesion or invasion of a host cell.Moreover, the differential binding and invasion profiles allow designand use of broad spectra or targeted inhibition of Als protein familymember activities. Additionally, functional fragments that conferbinding and/or invasive activity allow elimination of unwanted foreignprotein sequences, thus, increasing the efficacy of the Als familyprotein member vaccine or therapeutic inhibitor.

The nature of the pathogenesis of C. albicans by adherence toendothelial cells is discussed in U.S. Pat. No. 5,578,309 which isspecifically incorporated herein by reference in its entirety. For adescription of the ALS1 gene and characteristics thereof, including thecharacterization of the gene product as an adhesin see, Fu, Y., G. Rieg,W. A. Forizi, P. H. Belanger, J. E. J. Edwards, and S. G. Filler. 1998.Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiaeinduces adherence to endothelial and epithelial cells. Infect. Immun.66:1783-1786; Hoyer, L. L. 1997. Fu Y, Ibrahim A S, Sheppard D C, ChenY-C, French S W, Cutler J E, Filler S G, Edwards, J E, Jr. 2002. Candidaalbicans Als1p: an adhesin that is a downstream effector of the EFG1filamentation pathway. Molecular Microbiology 44:61-72. Sheppard D C,Yeaman M R, Welch W H, Phan Q T, Fu Y, Ibrahim A S, Filler S G, Zhang M,Waring A J, Edwards, Jr., J E 2004. Functional and Structural Diversityin the Als Protein Family of Candida albicans. Journal BiologicalChemistry. 279: 30480-30489. The ALS gene family of Candida albicans.International Society for Human and Animal Mycology Salsimorge, Italy:(Abstract); Hoyer, L. L., S. Scherer, A. R. Shatzman, and G. P. Livi.1995. Candida albicans ALSI: domains related to a Saccharonzycescerevisiae sexual agglutinin separated by a repeating motif. Mol.Microbiol. 15:39-54.

In this regard, the human fungal pathogen Candida albicans colonizes andinvades a wide range of host tissues. Adherence to host constituentsplays an important role in this process. Two members of the C. albicansAls protein family (Als1p and Als5p) have been found to mediateadherence and exemplify the binding, adhesion and cell invasionactivities of Als protein family members. As described herein, membersof the ALS gene family were cloned and expressed in S. cerevisiae tocharacterize their individual functions. Distinct Als proteins conferreddistinct adherence profiles to diverse host substrates. Using chimericAls5p-Als6p constructs, the regions mediating substrate-specificadherence were localized to the N-terminal domains in Als proteins. Inparticular, a subset of Als proteins also mediated endothelial cellinvasion, a previously unknown function of this family. Consistent withthese results, homology modeling revealed that Als members containanti-parallel β-sheet motifs interposed by extended regions, homologousto adhesions or invasins of the immunoglobulin superfamily. This findingwas confirmed using circular dichroism and Fourier transform infraredspectrometric analysis of the N-terminal domain of Als1p. Specificregions of amino acid hypervariability were found among the N-terminaldomains of Als proteins, and energy-based models predicted similaritiesand differences in the N-terminal domains that probably govern thediverse function of Als family members. Collectively, these resultsindicate that the structural and functional diversity within the Alsfamily provides C. albicans with an array of cell wall proteins capableof recognizing and interacting with a wide range of host constituentsduring infection.

The invention provides a vaccine having an isolated Als protein familymember having cell adhesion activity, or an immunogenic fragmentthereof, and an adjuvant in a pharmaceutically acceptable medium. Thevaccine can be an Als protein family member derived from a Candidaspecies such as Candida albicans, Candida krusei, Candida tropicalis,Candida glabrata or Candida, parapsilosis. The Als protein family membercan be, for example, Als1p, Als3p, Als5p, Als6p, Als7p and Als9p, or animmunogenic fragment thereof. All other Als protein family memberswithin an Candida species can similarly be employed as a vaccine of theinvention.

The present invention utilizes the gene product of C. albicansagglutinin like sequence protein family member as a vaccine to treat,prevent, or alleviate disseminated candidiasis. The vaccine is effectiveagainst different strains of C. albicans as well as against differentCandida species. The Als protein family member can be, for example,Als1p, Als3p, Als5p, Als6p, Als7p and Als9p. The invention exploits therole of the ALS gene products in the adherence of and invasion by C.albicans to endothelial and/or epithelial cells and the susceptibilityof the Als protein family member-expressed surface protein for use as avaccine to retard the pathogenesis of the organism.

Pursuant to this invention, an ALS family member gene encodes a surfaceadhesin that is selected as the target of an immunotherapeutic strategyagainst C. albicans. A demonstration that the expression product of theALS1 gene, the Als1p protein, has structural characteristics typical ofsurface proteins and is, in fact, expressed on the cell surface of C.albicans is one criterion for proteins that act as adhesins to hosttissues. The Als protein family members can be structurallycharacterized as having a signal peptide at the N-terminus, aglycosylphosphatidylinosine (GPI) anchorage sequence in the C-terminus,and a central region comprising repeats rich in threonine and serine.Also, Als protein family members have N—, and 0-glycosylation sites,typical of proteins that are expressed on the cell surface. Indirectimmunofluorescence using a monoclonal antibody directed against theN-terminus of ALs1p, for example, revealed that ALs1p is expressedduring the log phase of blastospores. This expression of ALs1p isincreased during hyphal formation and is localized to the junction wherethe hyphal element extends from the blastospores as indicated by thediffused surface staining. Furthermore, this monoclonal antibody blockedthe enhanced adherence of C. albicans overexpression mutant toendothelial cells, thereby establishing the principle for immunotherapyapplications using ALs1p. Functional characteristics as they relate tocell adhesion and invasion of other Als family members are describedfurther below in Example VI.

Thus, according to one aspect, the invention provides an Als familymember surface adhesion protein, designated, for example, Als1p, Als3p,Als5p, Als6p, Als7p and Als9p, or a functional fragment, conjugate oranalogue thereof, having useful properties when formulated in apharmaceutical composition and administered as a vaccine with or withoutan adjuvant. An Als protein family member, combination of two or moreAls protein family members or one or more functional fragments,analogues, conjugates or derivatives thereof, can be obtained from, forexample, Candida albicans. Similar adhesin or invasin molecules oranalogues or derivatives thereof can be of candidal origin and can beobtainable, for example, from species belonging to the genera Candida,for example Candida parapsilosis, Candida krusei, Candida glabrata andCandida tropicalis. A surface adhesin or invasin protein according tothe invention can be obtained in isolated or purified form, and thus,according to one embodiment of the invention a substantially pure Alsprotein family member Candida surface adhesin protein, or functionalfragment, immunogenic fragment, analogue, conjugate or derivativethereof, is formulated as a vaccine to cause an immune response in apatient to elicit an immune response against Candida and/or to blockadhesion of the organism to the endothelial cells. Fragments of Alsprotein family members that exhibit similar binding, adhesion orinvasion activity as an intact Als protein family member is referred toherein as a functional fragment. Fragments of Als protein family membersthat are capable of eliciting an antibody or cellular immune responseagainst a Candida species are referred to herein as an immunogenicfragment. Exemplary functional fragments include the N-terminalpolypeptide region of the Als protein family member described furtherbelow in Example VI. Exemplarily immogenic fragments include theN-terminal Als polypeptide region, the C-terminal Als polypeptide regionas well as any other Als fragment that is sufficient to generate anantibody, cellular or both an antibody and cellular immune response.Such immogenic fragments can be as small as about four amino acids andas large as the intact polypeptide as well as include all polypeptidelengths in between.

An analogue or derivative of the surface adhesion protein according tothe invention can be identified and further characterized by thecriteria described herein for an ALS family member gene and/or geneproduct. For example, a null mutant of the analogue or derivative wouldshare markedly reduced adhesion to endothelial cells compared tocontrols. Similarly, over-expression of the analogue or derivative in anappropriate model would show an increased adherence to endothelial cellscompared to controls and would be confirmed as a cell surface adhesin inaccord with the criteria described above. Also, antisera to an analogueor derivative can cross-react with anti-Als protein family memberantibodies and can exhibit increased survival times when administered ina mouse model of disseminated candidiasis as disclosed herein.

The invention also provides a method of treating or preventingdisseminated candidiasis. The method includes administering animmunogenic amount of a vaccine an isolated Als protein family memberhaving cell adhesion or invasion activity, or an immunogenic fragmentthereof, in a pharmaceutically acceptable medium. The vaccine can beadministered with or without an adjuvant. The Als protein family membercan be derived from different Candida strains as well as from differentCandida species such as Candida albicans, Candida krusei, Candidatropicalis, Candida glabrata and Candida, parapsilosis. An Als proteinfamily member used in the method of treating or prevention disseminatedcandidias includes Als1p, Als3p, Als5p, Als6p, Als7p and Als9p.

The effectiveness of the vaccines of the invention against differentCandida strains, different Candida species, other bacteria andinfectious agents and their wide range of immune activity are describedfurther below and exemplified in the Examples. For example, Example Vshows that anti-ALS antibodies are effective against mucosal andhematogenously disseminated candidal infections. Example VII shows thatvaccination with rAls1p-N improves survival during murine disseminatedcandidiasis by enhancing cell-mediated immunity. Example VIII shows thatthe vaccines of the invention reduce fungal burden and improve survivalin both immunocompetent and immunocompromised mice. Example IX shows theeffectiveness of the ALS vaccines of the invention against S. aureusinfections. Example X exemplifies that the vaccines of the invention areeffective against different strains of C. albicans and against differentspecies such as C. glabrata, C. krusei, C. parapsilosis and C.tropicalis as well as effectiveness in different animal models. ExampleXI also exemplifies the effectiveness of the different vaccines of theinvention in different animal models as well as provides a comparison ofthe different responses elicited and potency of two representative ALSvaccines.

The invention further provided is a method of treating or preventingdisseminated candidiasis that includes administering an effective amountof an isolated Als protein family member having cell adhesion activity,or an functional fragment thereof, to inhibit the binding or invasion ofCandida to a host cell or tissue. The Als protein family member can bederived from Candida albicans, Candida krusei, Candida tropicalis,Candida glabrata, and Candida, parapsilosis. An Als protein familymember used in the method of treating or prevention disseminatedcandidias includes Als1p, Als3p, Als5p, Als6p, Als7p and Als9p. The celladhesion activity includes binding to gelatin, fibronectin, laminin,epithelial cells or endothelial cells and/or promoting cell invasion.

In addition, the invention also provides a method of treating orpreventing Staphylococcus aureus infections using the Als protein familymembers described herein. In particular, the method of treating orpreventing Staphylococcus aureus infections includes administering animmunogenic amount of a vaccine an isolated Als protein family memberhaving cell adhesion activity, or an immunogenic fragment thereof, in apharmaceutically acceptable medium.

Als1p and Als3p are particularly efficacious because of significanthomology, to S. aureus cell surface proteins. The sequence andstructural homology of, for example, Als1p and Als3p, are describedfurther below in Example IX. Given the teachings and guidance providedherein, those skilled in the art will understand that the vaccines andmethods of the invention can be applied to the treatment of Candida andStaphylococcus infections alike. Similarly, given the teachings andmethods described herein, those skilled in the art also will understandthat the vaccines and methods of the invention also can be applied toother pathogens having cell surface polypeptides with similarimmunogenicity, sequence and/or structural homology to the Als proteinfamily members described herein, including fungus, bacteria and thelike.

Immunotherapeutic and/or Als polypeptide inhibition of cell adhesion orinvasion strategies against Candida or Staphylococcus infection canoperate at the level of binding to the vascular endothelial cells aswell as through a downstream effector of the filamentation regulatorypathway. An immunotherapeutic strategy or inhibition of binding using asoluble Als protein family member or functional fragment is useful inthis context because: (i) the morbidity and mortality associated withhematogenously disseminated candidiasis and other infectious pathogensremains unacceptably high, even with currently available antifungaltherapy; (ii) a rising incidence of antifungal and antibiotic resistanceis associated with the increasing use of antifungal and antibacterialagents, iii) the population of patients at risk for serious Candida andStaphylococcus infections is well-defined and very large, and includespost-operative patients, transplant patients, cancer patients and lowbirth weight infants; and iv) a high percentage of the patients whodevelop serious Candida infections are not neutropenic, and thus mayrespond to a vaccine or a competitive polypeptide or compound inhibitor.For these reasons, Candida and Staphylococcus are attractive fungal andbacterial targets for passive immunotherapy, active immunotherapy or acombination of passive or active immunotherapy. Additionally, Candidaalso is attractive for competitive inhibition using an Als proteinfamily member polypeptide, functional fragment thereof and/or a compoundor mimetic thereof that binds to one or more Als family members andprevents binding of Candida to a host cell receptor.

Given the teachings and guidance provided herein, those skilled in theart will understand that immunotherapeutic methods well know in the artcan be employed with the Als protein family members of the invention,immunogenic fragments, analogues, conjugates, and/or derivativesthereof, to use one or more of the molecule as an immunogen in apharmaceutically acceptable composition administered as a vaccine withor without an adjuvant. For the purposes of this invention, the terms“pharmaceutical” or “pharmaceutically acceptable” refer to compositionsformulated by known techniques to be non-toxic and, when desired, usedwith carriers or additives that can be safely administered to humans.Administration can be performed using well known routes including, forexample, intravenous, intramuscular, intraperitoneal or sub-cutaneousinjection. Such vaccines of the inventions also can include buffers,salts or other solvents known to these skilled in the art to preservethe activity of the vaccine in solution. Similarly, any of a wide rangeof adjuvants well known in the art can be employed with the vaccine ofthe invention to elicit, promote or enhance a therapeutically effectiveimmune response capable of reducing or blocking binding, invasion and/orinfection of Candida or Staphylococcus to a susceptible host cell.

Similarly, given the teachings and guidance provided herein, thoseskilled in the art also will understand that therapeutic methods wellknown in the art for administering and selectively blocking the bindingof cell surface molecules to their cognate receptors also can beemployed with the Als protein family members of the invention,functional fragments, analogues, conjugates and/or derivatives thereof,to use one or more of the Als protein family member as an inhibitor in apharmaceutically acceptable composition. As with vaccine formulations,inhibitory formulations can similarly be administered using well knownmethod in the art including, for example, intravenous intramuscular,intraperitoneal or sub-cutaneous injection. Such inhibitory compositionsthat bind Als family member receptors and block an Als protein familymember binding also can include buffers, salts or other solvents knownto these skilled in the art to preserve the activity of the vaccine insolution. Further, any of a wide range of formulations well known in theart can be employed with the inhibitory compositions of the invention totarget and/or enhance delivery or uptake so as to reduce or inhibitbinding, invasion and/or infection of Candida or Staphylococcus to asusceptible host cell.

With respect to the molecule used as a therapeutic immunogen or receptorbinding inhibitor pursuant to the present invention, those of skill inthe art will recognize that the Als protein family member molecules canbe truncated or fragmented without losing the essential qualities as animmunogenic vaccine or cell adhesion or invasion inhibitor. For example,an Als protein family member can be truncated to yield an N-terminalfragment by truncation from the C-terminal end with preservation of thefunctional properties described above and further below in the Examples.Similarly, C-terminal fragments can be generated by truncation from theN-terminal end with preservation of their functional properties. Othermodifications in accord with the teachings and guidance provided hereincan be made pursuant to this invention to create other Als proteinfamily member functional fragments, immunogenic fragments, analogs orderivatives thereof, to achieve the therapeutically useful propertiesdescribed herein with the native protein.

One aspect of the therapeutic effectiveness of Als protein familymembers and methods of the invention achieves interference withregulation of filamentation, to block adherence of the organism to hostconstituents, and to enhance clearance of the organism by immunoeffectorcells and other physiological mechanisms. Since endothelial cells coverthe majority of the vasculature, strategies to block the adherence,invasion and/or both of the organism to endothelial cells usingantibodies, Als family member proteins, polypeptide or peptides or anycombination thereof include useful embodiment of the present invention.As described previously, such adherence and/or invasion blockingtherapies include active or passive immunotherapy or inhibitory bindingdirected against the candidal adhesins, invasins, or cognate receptorsdisclosed herein. Thus, for example, any suitable host can be injectedwith protein and the serum collected to yield the desired anti-adhesinantibody after appropriate purification and/or concentration. Prior toinjection, the adhesin or invasin protein or a combination thereof, canbe formulated in a suitable vehicle preferably a known immunostimulantsuch as a polysaccharide or delivery formulation such as liposomes ortime-released compositions. Thus, according to a further aspect,invention provides a pharmaceutical composition comprising a candidaladhesin or invasin protein together with one or more pharmaceuticallyacceptable excipients in a formulation for use as a vaccine or Alsreceptor inhibitor.

The method of the invention is ameliorating and/or preventing candidalor Staphylococcus infection by blocking the adherence of C. albicans tothe endothelial or epithelial cells of a host constituent or by, forexample, antibody binding to the Staphylococcus and allowing immunemechanisms remove the pathogen. Thus, according to one aspect of theinvention, a pharmaceutical composition comprising an Als protein familymember adhesin or invasin protein, functional or immunogenic fragment,derivative, analogue, or conjugate thereof is formulated as a vaccine orAls receptor inhibitor in a pharmaceutical composition containing abiocompatible carrier for injection or infusion and is administered to apatient. Also, direct administration of antiserum raised against Alsfamily member protein or isolated or recombinant Als family memberprotein can be used to block the adherence of C. albicans to a mammalianhost constituent or effect the removal of a Staphylococcus pathogen.Antiserum against adhesin protein can be obtained by known techniques,Kohler and Milstein, Nature 256: 495-499 (1975), and may be humanized toreduce antigenicity, see U.S. Pat. No. 5,693,762, or produced intransgenic mice leaving an unrearranged human immunoglobulin gene, seeU.S. Pat. No. 5,877,397. Similarly, isolated or recombinant Als proteinfamily member also can be produced using methods well known to thoseskilled in the art including, for example, the recombinant productiondescribed in the Examples below.

A still further use of the invention, for example, is using the Alsprotein family member adhesin or invasin protein to develop vaccinestrategies for the prevention and/or amelioration of candidal orStaphylococcus infections. Thus, according to one aspect of theinvention, for example, standard immunology techniques can be employedto construct a multi-component vaccine strategy that can enhance and/orelicit immune response from a host constituent to bock adherence of C.albicans or to effect the elimination of Staphylococcus pathogens.

A still further use of the invention, for example, is developing DNAvaccine strategies. Thus, according to one aspect of the invention, forexample, the ALS family member polynucleotides encoding Als proteinfamily member adhesin or invasin or a functional fragment thereof isadministered according to a protocol designed to yield an immuneresponse to the gene product. See e.g., Feigner U.S. Pat. No. 5,703,055.

A still further use of the invention, for example, is developingcombination vaccine strategies. Thus, according to one aspect of theinvention, for example, anti-ALS protein family member antibodies may beused with antibodies in treating and/or preventing candidal orStaphylococcus infections. See U.S. Pat. No. 5,578,309.

The following Examples illustrate the immunotherapeutic utility of theALS1 adhesin as the basis for preventive measures or treatment ofdissemiated candidiasis. Example 1 describes the'preparation of an ALS1null mutant and a strain of C. albicans characterized by overexpressionof ALS1 to confirm the mediation of adherence to endothelial cells.Example 2 describes the localization of Als1p and the implication of theefg filamentation regulatory pathway. Example 3 describes thepurification of ALS1 adhesin protein. Example 4 describes thepreparation of rabbit polyclonal antibodies raised against the ALS1surface adhesin protein to be used to demonstrate the blocking of thesurface adhesin protein. Example 5, describes the blocking of adherencein vivo, using polyclonal antibodies raised against the ALS1 surfaceadhesion protein as described herein according to the invention toprotect against disseminated candidiasis in a mouse model. Example VIdescribes the structural and functional characteristics of Als proteinfamily members.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example 1 Als1 Mediates Adherence of C. albicans to Endothelial Cells

The URA blaster technique was used to construct a null mutant of Calbicans that lacks express of the Als1p. The als1/als1 mutant wasconstructed in C. albicans strain CAI4 using a modification of theUra-blaster methodology (Fonzi and Irwin, Genetics 134, 717 (1993)) asfollows: Two separate als1-hisG-IRA3-hisG-als1 constructs were utilizedto disrupt the two different alleles of the gene. A 4.9 kb AsLSI codingsequence was generated with high fidelity PCR (Boehringer Mannheim,Indianapolis, Ind.) using the primers:5′-CCCTCGAGATGCTTCAACAATTTACATTGTTA-3′ (SEQ ID NO:8) and5′-CCGCTCGAGTCACTAAATGAACAAGGACAATA-3′ (SEQ ID NO:9). Next, the PCRfragment was cloned into pGEM-T vector (Promega, Madison, Wis.), thusobtaining pGEM-T-ALS1. The hisG-URA3-hisG construct was released frompMG-7 by digestion with Kpn1 and Hind3 and used to replace the portionof ALS1 released by Kpn1 and Hind3 digestion of pGEM-T-ALS1. The finalals1-hisG-URA3-hisG-als1 construct was released from the plasmid bydigestion with Xhol and used to disrupt the first allele of ALS1 bytransformation of strain CAI-4.

A second als1-hisG-URA3-hisG-als1 construct was generated in two steps.First, a Bg12-Hind3 hisG-URA3-hisG fragment of pMB7 was cloned into theBamH1-Hind3 sites of pUC19, thereby generating pYC2. PYC2 was thendigested with Hind3, partially filled in with dATP and dGTP using T4 DNApolymerase, and then digested with Sma1 to produce a new hisGURA3-hisGfragment. Second, to generate ALS1 complementary flanking regions,pGEM-T-ALS1 was digested with Xbal and then partially filled in withdCTP and dTTP. This fragment was digested with Hpa1 to delete thecentral portion of ALS1 and then ligated to the hisG-URA3-hisG fragmentgenerating pYC3. This plasmid was then digested by Xhol to release aconstruct that was used to disrupt the second allele of the ALS1. Growthcurves were done throughout the experiment to ensure that the generatedmutations had no effect on growth rates. All integrations were confirmedby Southern blot analysis using a 0.9 kb ALS1 specific probe generatedby digestion of pYF5 with XbaI and HindIII.

The null mutant was compared to C. albicans CAI-12 (a URA+revertantstrain) for its ability to adhere in vitro to human umbilical veinendothelial cells. For adherence studies, yeast cells from YPD (2%glucose, 2% peptone, and 1% yeast extract) overnight culture, were grownin RPMI with glutamine at 25° C. for 1 hour to induce Als1p expression.3×10² organisms in Hanks balanced salt solution (HBSS) (IrvineScientific, Irvine, Calif.) were added to each well of endothelialcells, after which the plate was incubated at 37° C. for 30 minutes. Theinoculum size was confirmed by quantitative culturing in YPD agar. Atthe end of incubation period, the nonadherent organisms were aspiratedand the endothelial cell monolayers were rinsed twice with HBSS in astandardized manner. The wells were over laid with YPD agar and thenumber of adherent organisms were determined by colony counting.Statistical treatment was obtained by Wilcoxon rank sum test andcorrected for multiple comparisons with the Bonferroni correction.P<0.001.

Referring to FIG. 1, a comparison of the ALS1/ALS1 and als1/als1 strainshowed that the ALS1 null mutant was 35% less adherent to endothelialcells than C. albicans CAI-12. To reduce background adherence, theadherence of the wild-type strain grown under non-ALS1 expressingconditions was compared with a mutant autonomously expressing Als1p.This mutant was constructed by integrating a third copy of ALS1 underthe control of the constitutive ADH1 promoter into the wild-type C.albicans. To achieve constitutive expression of the ALS1 in C. albicans,a blunt-ended PCR generated URA3 gene is ligated into a blunt-edged Bg12site of pOCUS-2 vector (Novagen, Madison, Wis.), yielding pOU-2. A 2.4kb Not1-Stul fragment, which contained C. albicans alcohol dehydrogenasegene (ADH1) promoter and terminator (isolated from pLH-ADHpt, and kindlyprovided by A. Brown, Aberdeen, UK), was cloned into pOU-2 afterdigestion with Not1 and Stul. The new plasmid, named pOAU-3 had only oneBg12 site between the ADH1 promoter and terminator. ALS1 coding sequenceflanked by BamH1 restriction enzyme sites was generated by high fidelityPCR using pYF-5 as a template and the following primers:5′-CGGGATCCAGATGCTTCA-ACAATTTACATTG-3′ (SEQ ID NO:10) and5′-CGGGATCCTCACTAATGAACAAGGACAATA-3′ (SEQ ID NO:11). This PCR fragmentwas digested with BamH1 and then cloned into the compatible Bg12 site ofpOAU-3 to generate pAU-1. Finally, pAU-1 was linearized by XbaI prior totransforming C. albicans CAI-4. The site-directed integration wasconfirmed by Southern Blot analysis. Referring to FIG. 1B,overexpressing ALS1 in this P_(ADH1)-ALS1 strain resulted in a 76%increase in adherence to endothelial cells compared to the wild-type C.albicans. In comparing endothelial cell adherence of the wild-type tothat of the overexpressing mutant, yeast cells were grown overnight inYPD at 25° C. (non-inducing condition of Als1p). Als1p expression wasnot induced to reduce the background adherence of the wile-type, thusmagnifying the role of Als1p in adherence through P_(ADH1)-ALS1 hybridgene. The adherence assay was carried out as described above.Statistical treatment was obtained by Wilcoxon rank sum test andcorrected for multiple comparisons with the Bonferroni correction.P<0.001.

A monoclonal anti-Als1p murine IgG antibody was raised against apurified and truncated N-terminus of Als1p (amino acid #17 to #432)expressed using Clontech YEXpress™ Yeast Expression System (Palo Alto,Calif.). The adherence blocking capability of these monoclonalanti-Als1p antibodies was assessed by incubating C. albicans cells witheither anti-Als1 antibodies or mouse IgG (Sigma, St. Louis, Mo.) at a1:50 dilution. After which the yeast cells were used in the adherenceassay as described above. Statistical treatment was obtained by Wilcoxonrank sum test and corrected for multiple comparisons with the Bonferronicorrection. P<0.001. The results revealed that the adherence of theP_(ADH1)-ALS1 strain was reduced from 26.8%±3.5% to 14.7%±5.3%. Thus,the effects of ALS1 deletion and overexpression demonstrate that Als1pmediates adherence of C. albicans to endothelial cells.

Example II Localization of Als1p

For Als1p to function as an adhesin, it must be located on the cellsurface. The cell surface localization of Als1p was verified usingindirect immunofluorescence with the anti-Als1p monoclonal antibody.Diffuse staining was detected on the surface of blastospores duringexponential growth. This staining was undetectable on blastospores inthe stationary phase. Referring to FIG. 2A, when blastospores wereinduced to produce filaments, intense staining was observed thatlocalized exclusively to the base of the emerging filament. Noimmunofluorescence was observed with the als1/als1 mutant, confirmingthe specificity of this antibody for Als1p. See FIG. 2B. These resultsestablish that Als1p is a cell surface protein.

The specific localization of Als1p to the blastospore-filament junctionimplicates Als1p in the filamentation process. To determine themechanism, the filamentation phenotype of the C. albicans ALS1 mutantswas analyzed. Referring to FIG. 3A, the als1/als1 mutant failed to formfilaments after a 4 day incubation on Lee's solid medium, while both theALS1/ALS1 AND ALS1/als1 strains as well as the ALS1-complemented mutantproduced abundant filaments at this time point. The als1/als1 mutant wascapable of forming filaments after longer periods of incubation.Furthermore, overexpressing ALS1 augmented filamentation: theP_(ADH1-ALS)1 strain formed profuse filaments after a 3 day incubation,whereas the wild-type strain produced scant filaments at this timepoint. See FIG. 3B. To further confirm the role of Als1p infilamentation, a negative control was provided using mutant similar tothe ALS1 overexpression mutant, except the coding sequence of the ALS1was inserted in the opposite orientation. The filamentation phenotype ofthe resulting strain was shown to be similar to that of the wild-typestrain. The filament-inducing properties of Als1p are specific to cellsgrown on solid media, because all of the strains described abovefilamented comparably in liquid media. The data demonstrates that Als1ppromotes filamentation and implicates ALS1 expression in the regulationof filamentation control pathways. Northern blot analysis of ALS1expression in mutants with defects in each of these pathways, includingefg1/efg1, cph1/cph1, efg1/efg cph1/cph1, tup1/tup1, and cla4/cla4mutants were performed. Referring to FIG. 4A, mutants in Which bothalleles of EFG1 had been disrupted failed to express ALS1. Introductionof a copy of wild-type EFG1 into the efg1/efg1 mutant restored ALS1expression, though at a reduced level. See FIG. 4B. Also, as seen inFIG. 4A, none of the other filamentation regulatory mutationssignificantly altered ALS1 expression (FIG. 4A). Thus, Efg1p is requiredfor ALS1 expression.

If Efg1p stimulates the expression of ALS1, which in turn inducesfilamentation, the expression of ALS1 in the efg1/efg1 strain shouldrestore filamentation. A functional allele of ALS1 under the control ofthe ADH1 promoter was integrated into the efg1/efg1 strain. Toinvestigate the possibility that ALS1 gene product might complement thefilamentation defect in efg1 null mutant, an Ura efg1 null mutant wastransformed with linearized pAU-1. Ura⁺ clones were selected andintegration of the third copy of ALS1 was confirmed with PCR using theprimers: 5′-CCGTTTATACCATCCAATC-3′ (SEQ ID NO:13) and 5′-CTACATCCTCCAATGATATAAC-3′ (SEQ ID NO:14). The resulting strain expressed ALS1autonomously and regained the ability to filament on Lee's agar. SeeFIGS. 4B and C. Therefore, Efg1p induces filamentation throughactivation of ALS1 expression.

Because filamentation is a critical virulence factor in C. albicansdelineation of a pathway that regulates filamentation has importantimplications for pathogenicity. Prior to ALS1, no gene encoding adownstream effector of these regulatory pathways had been identified.Disruption of two other genes encoding cell surface proteins, HWP1 ANDINTI, results in mutants with filamentation defects. Although HWP1expression is also regulated by Efg1p, the autonomous expression of HWP1in the efg1/efg1 mutant fails to restore filamentation. Therefore Hwplpalone does not function as an effector of filamentation downstream ofEFG1. Also, the regulatory elements controlling INT1 expression are notknow. Thus, Als1p is the first cell-surface protein identified thatfunctions as a downstream effector of filamentation, thereby suggestinga pivotal role for this protein in the virulence of C. albicans.

The contribution of Als1p to C. albicans virulence was tested in a modelof hematogenously disseminated candidiasis, A. S. Ibrahim et al.,Infect. Immun. 63, 1993 (1995). Referring to FIG. 5A, mice infected withthe als1/als1 null mutant survived significantly longer than miceinfected with the ALS1/ALS1 strain, the ALS1/als1 mutant or theALS1-complemented mutant. After 28 hours of infection, the kidneys ofmice infected with the als1/als1 mutant contained significantly fewerorganisms (5.70±0.46 log₁₀ CFU/g) (P<0.0006 for both comparisons). Nodifference was detected in colony counts of organisms recovered fromspleen, lungs, or liver of mice infected with either of the strains atany of the tested time points. These results indicate that Als1p isimportant for C. albicans growth and persistence in the kidney duringthe first 28 hours of infection. Referring to FIG. 5B, examination ofthe kidneys of mice after 28 hours of infection revealed that theals1/als1 mutant produced significantly shorter filaments and elicited aweaker inflammatory response than did either the wild-type ofALS1-complemented strains. However, by 40 hours of infection, the lengthof the filaments and the number of leukocytes surrounding them weresimilar for all three strains.

The filamentation defect of the als1/als1 mutant seen on histopathologyparalleled the in vitro filamentation assays on solid media. This mutantshowed defective filamentation at early time points both in vivo and invitro. This defect eventually resolved with prolongedinfection/incubation. These results suggest that a filamentationregulatory pathway that is independent of ALS1 may become operative atlater time points. The activation of this alternative filamentationpathway by 40 hours of infection is likely the reason why mice infectedwith the als1/als1 mutant subsequently succumbed in the ensuing 2-3days.

Collectively, these data demonstrate that C. albicans ALS1 encodes acell surface protein that mediates both adherence to endothelial cellsand filamentation. Als1p is the only identified downstream effector ofany known filamentation regulatory pathway in C. albicans. Additionally,Als1p contributes to virulence in hematogenous candidal infection. Thecell surface location and dual functionality of Als1p make it anattractive target for both drug and immune-based therapies.

Example III Purification of ALS1 Adhesin Protein

The ALS1 protein synthesized by E. coli is adequate as an immunogen.However eukaryotic proteins synthesized by E. coli may not be functionaldue to improper folding or lack of glycosylation. Therefore, todetermine if the ALS1 protein can block the adherence of C. albicans toendothelial cells, the protein is, preferably, purified from geneticallyengineered C. albicans.

PCR was used to amplify a fragment of ALS1, from nucleotides 52 to 1296.This 1246 bp fragment encompassed the N-terminus of the predicted ALS1protein from the end of the signal peptide to the beginning of thetandem repeats. This region of ALS1 was amplified because it likelyencodes the binding site of the adhesin, based on its homology to thebinding region of the S. cerevisiae Aga1 gene product. In addition, thisportion of the predicted ALS1 protein has few glycosylation sites andits size is appropriate for efficient expression in E. coli.

The fragment of ALS1 was ligated into pQE32 to produce pINS5. In thisplasmid, the protein is expressed under control of the lac promoter andit has a 6-hits tag fused to its N-terminus so that it can be affinitypurified. We transformed E. coli with pINS5, grew it under inducingconditions (in the presence of IPTG), and then lysed the cells. The celllysate was passed through a Ni²⁺-agarose column to affinity purify theALS1-6His fusion protein. This procedure yielded substantial amounts ofALS1-6His. The fusion protein was further purified by SDS-PAGE. The bandcontaining the protein was excised from the gel so that polyclonalrabbit antiserum can be raised against it. It will be appreciated by oneskilled in the art that the surface adhesin protein according to theinvention may be prepared and purified by a variety of known processeswithout departing from the spirit of the present invention. The sequenceof Als1p is listed in FIG. 7.

Example IV Raising Polyclonal Antisera Against Als1 Protein

To determine whether antibodies against the ALS1 protein block theadherence of Candida albicans to endothelial and epithelial cells, andthe selected host constituent in vitro, rabbits were inoculated with S.cerevisiae transformed with ALS1 protein. The immunization protocol usedwas the dose and schedule used by Hasenclever and Mitchell forproduction of antisera that identified the antigenic relationship amongvarious species of Candida. Hasenclever, H. F. and W. O. Mitchell. 1960.Antigenic relationships of Torulopsis glabrata and seven species of thegenus Candida. J. Bacteriol. 79:677-681. Control antisera were alsoraised against S. cerevisiae transformed with the empty plasmid. Allyeast cells were be grown in galactose to induce expression of the ALSgenes. Before being tested in the adherence experiments, the serum washeat-inactivated at 56 C to remove all complement activity.

Sera from immunized rabbits were absorbed with whole cells of S.cerevisiae transformed with empty plasmid to remove antibodies that arereactive with components of the yeast other than ALS1 protein. The titerof the antisera was determined by immunofluorescence using S. cerevisiaethat express the ALS1 gene. FITC-labeled anti-rabbit antibodies werepurchased from commercial sources (Southern Biotechnology, Inc).Affinity-purified secondary antibodies were essential because manycommercially available sera contain antibodies reactive with yeastglucan and mannan. The secondary antibodies were pretested using Candidaalbicans as well as S. cerevisiae transformed with the plasmid and wereabsorbed as needed to remove any anti-S. cerevisiae or anti-Candidaantibodies. Negative controls were 1) preimmune serum 2) S. cerevisiaetransformed with the empty plasmid, and 3) S. cerevisiae transformedwith the ALS gene but grown under conditions that suppress expression ofthe ALS gene (glucose).

In addition to the above experiments, Western blotting was used toprovide further confirmation that an antiserum binds specifically to theALS protein against which it was raised. S. cerevisiae transformed withthe ALS1 were grown under inducing conditions and their plasma membraneswere isolated by standard methods. Panaretou R and P. Piper. 1996.Isolation of yeast plasma membranes. p. 117-121. In I. H. Evans. (ed.),Yeast Protocols. Methods in Cell and Molecular Biology. Humana Press,Totowa, N.J. Plasma membranes were also prepared from S. cerevisiaetransformed with the empty plasmid and grown under identical conditions.The membrane proteins were separated by SDS-PAGE and then transferred toPVDF membrane by electroblotting. Harlow, E. and D. Lane. 1988.Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press.After being blocked with nonfat milk, the blot was incubated with theALS antiseru. The preabsorbed antiserum did not react with proteinsextracted from S. cerevisiae containing empty plasmid. This antiserumblocked the adherence of S. cerevisiae pYF5 (a clone that expressesCandida albicans ALS1) to endothelial cells.

Example V Polyclonal Antibodies Against Specific ALS ProteinsProphylactically Protect Mice from Mucosal and HematogenouslyDisseminated Candidal Infections

Having identified the antisera that block the adherence of a clone of S.cerevisiae transformed with an ALS gene under the above conditions,these antisera were demonstrated to protect mice from intravenouschallenge with Candida albicans.

The antisera against the ALS proteins were first tested in the murinemodel of hematogenously disseminated candidiasis. Affinity-purifiedanti-ALS antibodies are effective in preventing adhesion of yeast cellsto various substrates (see EXAMPLE 3). Affinity-purification is usefulin this system because antibody doses can be accurately determined.Moreover, the unfractionated antisera will undoubtedly contain largeamounts of antibody directed toward antigens on the S. cerevisiaecarrier cells. Many of these anti-Saccharomyces antibodies would likelybind to C. albicans and make interpretation of the results impossible.Additionally, it is quite possible that the procedure used to eluteantibodies from S. cerevisiae that express the ALS protein may alsoelute small amounts of yeast mannan or glucan that could haveadjuvant-like activity. The immunoaffinity-purified antibodies arefurther purified before use. They may also be preabsorbed with mousesplenocytes.

Antibody doses may be administered to cover the range that brackets thelevels of serum antibody that can be expected in most activeimmunization protocols and to cover the range of antibody doses that aretypically used for passive immunization in murine models of candidiasis.See Dromer, F., J. Charreire, A. Contrepois, C. Carbon, and P. Yeni.1987, Protection, of mice against experimental cryptococcosis byanti-Cryptococcus neoformwns monoclonal antibody, Infect. Immun.55:749-752; Han, Y. and J. E. Cutler. 1995, Antibody response thatprotects against disseminated candidiasis, Infect. Immun. 63:2714-2719;Mukherjee, J., M. D. Scharff, and A. Casadevall. 1992, Protective murinemonoclonal antibodies to Cryptococcus neoformwns, Infect. Immun.60:4534-4541; Sanford, J. E., D. M. Lupan, A. M. Schlageter, and T. R.Kozel. 1990, Passive immunication against Cryptococcus neoformans withan isotype-switch family of monoclonal antibodies reactive withcryptococcal polysaccharide, Infect. Immun. 58:1919-1923. BALB/c Mice(femal, 7 week old, the NCI) were given anti-ALS that had been absorbedwith mouse splenic cells by an intraperitoneal (i.p.) injection. Controlmice received prebled serum that had been absorbed with mouse speniccells, intact anti-ALS serum, or DPBS, respectively. For thepre-absorption, 2 ml of anti-ALS or prebled sera were mixed with 100 μlof mouse (BALB/c, 7 weeks old female, NCI) splenic cells (app. 9×10⁶cells per ml) at room temperature for 20 minutes. The mixture was washedwith warm sterile DPBS by centrifugation (@300×g) for 3 minutes. Thisprocedure was repeated three times. The volume of i.p. injection was 0.4ml per mouse. Four hours later, the mice were challenged with C.albicans (strain CA-1; 5×10⁵ hydrophilic yeast cells per mouse by i.v.injection. Then, their survival times were measured. See FIG. 6.

Previous studies have shown that antibodies administered via theintraperitoneal route are rapidly (within minutes) and almost completelytransferred to the serum (Kozel and Casadevall, unpublishedobservations). As a control for effects of administering the antibodypreparations, a parallel group of mice were treated with antibodiesisolated from pre-immune serum that has been absorbed with S. cerevisiatransformed with the ALS gene. The survival time and numbers of yeastper gram of kidney were measured. Again, referring to FIG. 6, miceinfected intravenously with 10⁶ blastopores of ALS1 null mutant had alonger median survival time when compared to mice infected with Candidaalbicans CAI-12 or Candid albicans in which one allele of the ALS1 hadbeen deleted (p=0.003).

These results indicate that immunotherapeutic strategies using the ALS1proteins as a vaccine have a protective prophylactic effect againstdisseminated candidiasis.

Example VI Functional and Structural Diversity in the Als Protein Familyof Candida albicans

Isolation and characterized of the C. albicans ALS1 gene by heterologouscomplementation of nonadherent S. cerevisiae has been previouslydescribed (Fu et al., Infect. Immun. 66:1783-1786 (1998)). ALS1 encodesa cell surface protein that mediates adherence to endothelial andepithelial cells. Disruption of both copies of this gene in C. albicansis associated with a 35% reduction in adherence to endothelial cells,and overexpression of ALS1 increases adherence by 125% (Fu et al., Mol.Microbiol. 44:61-72 (2002)).

ALS1 is a member of a large C. albicans gene family consisting of atleast eight members originally described by Hoyer et al. (Hoyer et al.,Trends Microbiol. 9:176-180 (2001), Zhao et al., Microbiology149:2947-2960 (2003)). These genes encode cell surface proteins that arecharacterized by three domains. The N-terminal region contains aputative signal peptide and is relatively conserved among Als proteins.This region is predicted to be poorly glycosylated (Zhao et al.,Microbiology 149:2947-2960 (2003), Hoyer et al., Genetics 157:1555-1567(2001)). The central portion of these proteins consists of a variablenumber of tandem repeats (˜36 amino acids in length) and is followed bya serine-threonine-rich C-terminal region that contains aglycosylphosphatidylinositol anchor sequence (supra). Whereas theproteins encoded by this gene family are known to be expressed duringinfection (Hoyer et al., Infect. Immun. 67:4251-4255 (1999), Zhang etal., Genome Res. 13:2005-2017 (2003)), the function of the different Alsproteins has not been investigated in detail.

Heterologous expression of Als proteins in nonadherent S. cerevisiae wasperformed to evaluate the function of Als proteins and to avoid the highbackground adherence mediated by the multiple other adhesins expressedby C. albicans. This heterologous expression system has been usedextensively for the study of C. albicans genes, including the isolationand characterization of the adhesins ALS1, ALS5, and EAP1 (Li et al.,Eukaryot Cell 2:1266-1273 (2003), Fu et al, Infect. Immun. 66:1783-1786(1998), Gaur et al., Infect. Immun. 65:5289-5294 (1997)). As describedfurther below, using this model system Als proteins were demonstrated tohave diverse adhesive and invasive functions. Consistent with theseresults, homology modeling indicated that Als proteins are closelyrelated in structure to adhesin and invasin members of theimmunoglobulin superfamily of proteins. Structural analyses using CD andFourier transform infrared (FTIR) 1 spectrometry confirmed that theN-terminal domain of Als1p is composed of anti-parallel β sheet, turn,α-helical, and unstructured domains consistent with the structures ofother members of the immunoglobulin superfamily. Finally, comparativeenergy-based models suggest differences in key physicochemicalproperties of the N-terminal domains among different Als proteins thatmay govern their distinct adherence and invasive biological functions.

To clone ALS family members and express them in S. cerevisiae, ALS1, -3,-5, -6, -7, and -9 were successfully amplified and expressed asdescribed below. Briefly, for cloning and other culture steps, S.cerevisiae strain S150-2B (leu2 his3 trp1 ura3) was used forheterologous expression as has been described previously (Fu et al.,Infect. Immun. 66:2078-2084 (1998)). C. albicans strain SC5314 was usedfor genomic cloning. All strains were grown in minimal defined medium(1× yeast nitrogen base broth (Difco), 2% glucose, and 0.5% ammoniumsulfate, supplemented with 100 μg/ml L-leucine, -L tryptophan,L-histidine, and adenine sulfate) solidified with 1.5% bacto-agar(Difco) as needed. Growth of ura-strains was supported by the additionof 80 μg/ml uridine (Sigma). Plasmids pGK103, containing ALS5, pYF5,containing ALS1, and pALSn, containing ALS9, have been describedpreviously (Fu et al., Infect. Immune. 66:1783-1786 (1998), Gaur et al.,Infect. Immune. 65:5289-5297 (1997), Lucinod et al., Proceedings of the102nd Annual Meeting of the American Society for Microbiology, pp. 204,American Society for Microbiology, Salt Lake City, Utah. (2002)).Plasmid pADH1, obtained from A. Brown (Aberdeen, UK) contains the C.albicans alcohol dehydrogenase gene (ADH1) promoter and terminator,which are functional in S. cerevisiae (Bailey et al., J. Bacteriol.178:5353-5360 (1996)). This plasmid was used for constitutive expressionof ALS genes in S. cerevisiae.

Human oral epithelial and vascular endothelial cells were obtained andcultured as follows. The FaDu oral epithelial cell line, isolated from apharyngeal carcinoma, was purchased from the American Type CultureCollection (ATCC) and maintained as per their recommended protocol.Endothelial cells were isolated from umbilical cord veins and maintainedby our previously described modification of the method of Jaffe et al.(Fu et al., Mol. Microbiol. 44:61-72 (2002), Jaffe et al., J. Clin.Invest. 52:2745-2756 (1973)). All cell cultures were maintained at 37°C. in a humidified environment containing 5% CO2.

For cloning the ALS genes, genomic sequences of members of the ALSfamily were identified by BLAST searching of the Stanford data base(available on the World Wide Web at URL:sequence.stanford.edu/group/candida/search.html). PCR primers weregenerated to specifically amplify each of the open reading frames thatincorporated a 5′ BglII and a 3′ XhoI restriction enzyme site and areshown below in Table I (SEQ ID NOS:14-19 (ALS 1, 3, 5, 6, 7 and 9 senseprimers, respectively); SEQ ID NOS:20-25 ((ALS 1, 3, 5, 6, 7 and 9antisense primers, respectively)). Each gene was cloned by PCR using theExpand® High Fidelity PCR system (Roche Applied Science). ALS3, ALS6,and ALS7 were amplified from C. albicans SC5314 genomic DNA, whereasALS1, ALS5, and ALS9 were amplified from plasmids that had beenpreviously retrieved from C. albicans genomic libraries (Fu et al.,Infect. Immune. 66:1783-1786 (1998), Gaur et al., Infect. Immune.65:5289-5297 (1997), Lucinod et al., Proceedings of the 102nd AnnualMeeting of the American Society for Microbiology, pp. 204, AmericanSociety for Microbiology, Salt Lake City, Utah. (2002)). PCR productswere ligated into pGEM-T-Easy (Promega) for sequencing.Sequence-verified ALS open reading frames were then released frompGEM-T-Easy by BglII-XhoI co-digestion and ligated into pADH1, such thatthe ALS gene of interest was under the control of the ADH1 promoter. S.cerevisiae strain S150-2B was transformed with each of the ALSoverexpression constructs as well as the empty pADH1 construct using thelithium acetate method. Expression of each ALS gene in S. cerevisiae wasverified by Northern blot analysis before phenotypic analyses wereperformed.

TABLE I PCR primers used to amplify the coding regions ofALS gene for heterologous expression in S. cerevisia ALS geneSense (5′-3′) Antisense (5′-3′) ALS1 AGATCTCAGATGCTTCAACAATTTACTCGAGTCACTAAATGAA CATTG CAAGGACAATA ALS3 GAAGATCTATGCTACAACAATATACCCGCTCGAGTTAAATAAA ATTGTTACTC CAAGGATAATAATGTGATC ALS5AGATCTCAACTACCAACTGCTAACA CTCGAGACCATATTATTTG GTACAATC ALS6AGATCTCATTCACCGACAATGAAGA CTCGAGTTGGTACAATCCC CA GTTTGA ALS7AGATCTTCAACAGTCTAATACCTAT CTCGAGACTTGATTGAATT GA ATACCATATA ALS9AGATCTCGAATGCTACCACAATTCC CTCGAGTCTTAGCACCCTG TA ACGTAGCT

ALS mRNA expression was detected by Northern blot analysis for eachconstruct. Despite the use of three sets of primers, amplification ofALS2 and ALS4 from genomic DNA of C. albicans SC5314 was unsuccessful.Given the difficulty of sequencing and assembling across the tandemrepeats of ALS genes, it is possible that this outcome reflects errorsin the sequence assembly currently available on the published genomedata base.

Flow cytometry confirmed that each of the Als proteins was expressed onthe surface of their respective S. cerevisiae hosts. Briefly,confirmation of cell surface expression for each of the Als constructswas determined using indirect immunofluorescence employing two differentpolyclonal anti-Als antisera. Antiserum A consisted of anti-Als1pantibodies, generated by immunization of rabbits with a 417-amino acidN-terminal fragment of Als1p. Antiserum B was rabbit anti-C. albicansmannan factor 5 that recognizes C. albicans cell wall components butdoes not cross-react with S. cerevisiae (Iatron Laboratories).

For each strain, 10⁷ blastospores were isolated from overnight culture,blocked with 100 μl of goat serum, and then stained with eitherpolyclonal antiserum A or B at a 1:25 dilution, followed by fluoresceinisothiocyanate-labeled goat anti-rabbit IgG at 1:100. A FACSCaliber(Becton Dickinson) instrument equipped with an argon laser emitting at488 nm was used for flow cytometric analyses. Fluorescence emission wasdetected with a 515/40-nm bandpass filter. Fluorescence data for 10,000events were collected, and the distribution of cells with fluorescenceabove base line (i.e. S. cerevisiae transformed with the empty plasmid)was analyzed for each strain using CELLQUEST software (BectonDickinson).

As shown in Table II, two distinct antisera demonstrated that all of theAlsp-expressing strains exhibited at least a 4-fold increase influorescence when compared with S. cerevisiae transformed with the emptyplasmid. Consistent with the predicted structural diversity amongmembers of the Als family, the antisera displayed differences inrecognition of individual Als expression strains.

TABLE II Detection of Als proteins on the surface of S. cerevisiae byflow cytometric analysis Percentage of cells above background (-foldincrease) Als construct Antiserum A Antiserum B Empty plasmid (1) (1)Als1p 47.8 (17) 50.1 (19) Als3p 24.5 (9) 54.0 (20) Als5p 23.5 (8) 28.2(11) Als6p 12.7 (4) 16.2 (6) Als7p 22.1 (8) 15.7 (6) Als9p 11.4 (4) 33.9(13) Blastospores of each strain were stained using indirectimmunofluorescence with either polyclonal anti-Als1p antiserum (A) orpolyclonal anti-C. albicans cell wall antiserum (B) and then analyzedusing flow cytometry. Restuls are expressed as percentage of positivecells above background (S. cerevisiae transformed with empty plasmid),with -fold increase in parentheses.

S. cerevisiae clones that expressed the various Als proteins wereexamined for their ability to adhere to a variety of host substrates. Asdescribed below, the results show that Als proteins display differentprofiles of substrate-specific adherence.

Fungal adherence assays were preformed to determine the adherenceproperties of transformed S. cerevisiae strains. Briefly, a modificationof previously described adherence assay (8) was employed as follows.Adherence plates were coated by adding 1 ml of a 0.01 mg/ml solution ofgelatin (Sigma), laminin (Sigma), or fibronectin (Becton Dickinson) toeach well of a 6-well tissue culture plate (Costar) and incubatingovernight at 37° C. For endothelial cells, second passage cells weregrown to confluence in 6-well tissue culture plates coated with a 0.2%gelatin matrix, and for epithelial cells, FaDU cells were grown toconfluence (3 days) in E-well tissue culture plates coated with a 0.1%fibronectin matrix. Before adherence testing, wells were washed twicewith 1 ml of warm Hanks' balanced salt solution (HBSS). S. cerevisiaestrains to be tested were grown overnight in minimal defined media at30° C. and then harvested by centrifugation, washed with HBSS (IrvineScientific), and enumerated using a hemacytometer. Three hundredorganisms were added to each well of a 6-well tissue culture platecoated with the substrate of interest and incubated for 30 min at 37° C.in CO₂. Nonadherent organisms were removed by washing twice in astandardized manner with 10 ml of HBSS. The wells were overlaid with YPDagar (1% yeast extract (Difco), 2% bacto-peptone (Difco), 2% D-glucose,1.5% agar), and the inoculum was confirmed by quantitative culture.Plates were incubated for 48 h at 30° C., and the colonies were counted.Adherence was expressed as a percentage of the initial inoculum.Differences in adherence were compared using a single factor analysis ofvariance test, with p<0.01 considered significant.

There were striking differences in the adherence profiles of the S.cerevisiae transformants to the different substrates (FIG. 8). WhereasAls1p-, Als3p-, and Als5p-expressing strains bound to all substratestested, Als6p-expressing S. cerevisiae adhered only to gelatin, andAls9p-expressing S. cerevisiae adhered above background levels only tolaminin. Further, there were quantitative differences in adherence tothe various substrates. For example, when compared with Als3p, Als1pconferred greater adherence to gelatin but less adherence to epithelialcells (p<0.01, single factor analysis of variance). Only S. cerevisiaeexpressing Als7p adhered to none of the substrates tested. Whereas smalldifferences in levels of Als protein expression cannot be ruled out bythe immunofluorescence studies shown in Table II, such differences areunlikely to be responsible for the substrate-specific binding patternsfound in this study. Such a global increase or decrease in the amount ofAls protein expressed on the cell surface would be expected to produce acommensurate increase or decrease in adherence across all substrates andnot result in the substrate-specific differences that were observed.

As described below, the substrate binding specificity for Als proteinsresides in the N-terminal sequences of Als Proteins. Briefly, Als5pexpression in S. cerevisiae confered adherence to multiple substrates,including gelatin and endothelial cells, whereas Als6p expressionresulted in adherence to gelatin alone. Despite this marked differencein function, Als5p and Als6p are more than 80% identical at the aminoacid level. The tandem repeat and C-terminal portions of these proteinsare virtually identical, and the majority of the sequence differencesare concentrated in the N termini of these two proteins. These dataindicate that N-terminal sequence variability confers substratespecificity.

The above result was supported by the results of studies determining theadherence phenotypes of chimeric ALS5/ALS6 constructs. Briefly, chimericAls5/Als6 proteins were constructed by exchanging the N termini of eachprotein. Chimeric ALS5/6 genes were constructed as follows. A BglII-HpaIfragment of ALS5 encompassing the 5′ 2117 bp of the gene was isolated.pGEM-T-ALS6 was then digested with BglII and HpaI to release thecorresponding 5′ 2126 bp of ALS6, and the fragment consisting ofpGEM-T-Easy plus the 3′ sequences of ALS6 was isolated and ligated tothe 5′ ALS5 fragment to generate plasmid pGEM-T-5N6C. An identicalapproach using the corresponding 5′ fragment of ALS6 and 3′ fragment ofALS5 was used to generate plasmid p-GEM-T-6N5C. After sequenceconfirmation, each chimeric ALS gene was released by BglII-XhoIdigestion and subcloned into pADH1 as above. S. cerevisiae S150-2B wasthen transformed with these constructs, and expression was verified byNorthern blot analysis before characterization of their adherenceproperties.

S. cerevisiae expressing a chimeric fusion of the N terminus of Als5p tothe C terminus of Als6p adhered to both gelatin and endothelial Cells ina manner similar to Als5p (FIG. 9). Likewise, strains expressing thechimeric fusion of the Als6 N terminus to the C terminus of Als5padhered only to gelatin, as did S. cerevisiae expressing Als6p (FIG. 9).Further, strains expressing Als5p and chimeric Als5N6C proteinagglutinated fibronectin-coated beads, whereas those expressing Als6pand chimeric Als6N5C protein had little to no affinity for these beads.Collectively, these data indicate that the adherence profiles of thesetransformed S. cerevisiae strains were governed by the N-terminalportion of the Als protein.

In addition to the differences in substrate specificity demonstratedbetween the Als protein family members, differences in other biologicalfunctions also were observed. For example, a subset of Als proteins wasshown to mediate endothelial cell invasion by S. cerevisiae. C. albicansinvades endothelial cells by inducing its own endocytosis (Filler etal., Infect. Immun. 63976-983 (1995), Belanger et al., Cell Microbiol.,in press (2002)). This endocytosis occurs after the organism adheres toendothelial cells; however, the C. albicans ligands required for thisprocess are unknown. Further, it is unclear if distinct candidal ligandsare required for both adherence and endocytosis. In addition to beingnonadherent, S. cerevisiae does not undergo significant endocytosis byendothelial cells. Therefore, to test whether Als proteins could serveas invasins as well as adhesins, the ability of S. cerevisiae strainsexpressing Als proteins to invade endothelial cells was determined.

The ability of Als proteins to mediate endothelial cell invasion wasdetermined using a modification of a previously described differentialfluorescence assay (Phan et al., Infect. Immun. 68:3485-3490 (2000)).Briefly, endothelial cells were grown to confluence on 12-mm diameterglass coverslips coated with fibronectin and placed in a 24-well tissueculture plate (Corning). Cells were then infected with 10⁵ blastosporesof each S. cerevisiae strain in RPMI 1640 medium (Irvine Scientific). Asa positive control, cells were infected with a similar number of C.albicans blastospores. After incubation for 90 min, the cells wererinsed twice with 0.5 ml of HBSS in a standardized manner and fixed with3% paraformaldehyde. Organisms remaining adherent to the surface of theendothelial cells were stained for 1 h with the rabbit anti-C. albicansantiserum (Biodesign), which had been conjugated with Alexa 568(Molecular Probes, Inc., Eugene, Oreg.), which fluoresces red. Thisantiserum cross-reacts with S. cerevisiae at a 2-fold higher dilution.The endothelial cells were then permeabilized in 0.2% Triton X-100 inphosphate-buffered saline for 10 min, after which the cell-associatedorganisms (the internalized plus adherent organisms) were again stainedwith the anti-C. albicans antiserum conjugated with Alexa 488, whichfluoresces green. The coverslips were then observed underepifluorescence. The number of organisms that had been internalized bythe endothelial cells was determined by subtracting the number ofadherent organisms (fluorescing red) from the number of cell-associatedorganisms (fluorescing green). At least 100 organisms were counted oneach coverslip, and all experiments were performed in triplicate on atleast three separate occasions.

Fibronectin bead adherence assays also was performed to furthercharacterize the binding characteristics of certain Als proteins. Inthis regard, Als5p was originally identified by virtue of the protein'sability to induce agglutination of fibronectin-coated beads whenexpressed on the surface of S. cerevisiae (Gaur et al., Infect. Immune.65:5289-5297 (1997)). Therefore, S. cerevisiae strains transformed withALS5, ALS6, 5N6C, and 6N5C for fibronectin were tested for beadadherence using this methodology (Gaur et al., Infect. Immune.65:5289-5297 (1997), Gaur et al., Infect. Immun. 67:6040-6047 (1999)).Briefly, tosylated magnetic beads (Dynal Biotech) were coated withfibronectin following the manufacturer's instructions. Next, 10 μl ofcoated beads (10⁶ beads) were mixed with 1×10⁸ transformed S. cerevisiaein 1 ml of 1× Tris-EDTA (TE) buffer, pH 7.0, and incubated with gentlemixing for 45 min. The tubes were placed in a magnet to separate beadsand adherent S. cerevisiae from nonadherent organisms. The supernatantcontaining nonadherent organisms was removed by aspiration, and theremaining beads were washed three times by resuspending in 1 ml of TEbuffer, followed by magnetic separation and aspiration of thesupernatant. Finally, the washed beads and adherent organisms wereresuspended in 100 μl of TE buffer and examined microscopically forco-agglutination.

The results show that S. cerevisiae expressing Als1p, Als3p, and Als5pdisplayed a significant increase in the percentage of cell-associatedorganisms, reflecting their ability to adhere to endothelial cells. Inaddition, organisms expressing Als3p and, to a lesser extent, Als1p andAls1p demonstrated significant endothelial cell invasion (FIG. 10).

In addition to the functional studies described above, Als proteins alsowere found to be homologous to adhesins and invasins of theimmunoglobulin superfamily. As an initial step in the molecular modelingof Als proteins, a knowledge-based search algorithm was used to identifymolecules that share significant structural similarity with Als familymembers. Briefly, homology and energy-based modeling was conducted tocompare overall physicochemical features of Als proteins. First, aknowledge-based method (SWISS-MODEL) (Guex et al., Electrophoresis18:2714-2723 (1997), Schwede et al., Nucleic Acids Res. 31:3381-3385(2003)) was used to analyze and compare combinatorial extensionstructural alignments of structures in the Swiss and Brookhaven proteindata bases for proteins with homologous conformation (Shindyalov et al.,Protein Eng. 11:739-747 (1998)). This approach included the BLASTP2algorithm (Altschul et al., Mol. Biol. 215:403-410 (1990)) to search forprimary sequence similarities in the ExNRL-3D data base. In parallel,the dynamic sequence alignment algorithm SIM (Huang et al., Adv. Appl.Math. 12:337-367 (1991)) was used to select candidate templates withgreatest sequence identity. Subsequently, ProModII was used to conductprimary and refined match analyses. Resulting proteins were used astemplates for homology modeling of Als protein backbone trajectories.

Robust models of the N-terminal domains of Als proteins (e.g. aminoacids 1-480; preceding initial tandem repeats) were generated throughcomplementary approaches. The N-terminal domains of Als proteins wereconverted to putative solution conformations by sequence homology(Composer (Topham et al. Biochem. Soc. Symp. 57:1-9 (1990)) andthreading methods (Matchmaker (Godzik et al., J. Mol. Biol. 227:227-238(1992)) and Gene-Fold (Jaroszewski et al., Protein Sci. 7:1431-1440(1998), Godzik et al., Protein Eng. 8:409-416 (1995), Godzik et al.,Proc. Natl. Acad. Sci. U.S.A. 89:12098-12102 (1992), Godzik et al., J.Comput. Aided Mol. Des. 7:397-438 (1993)) using SYBYL 6.9.1 software(Tripos Associates) operating on Silicon Graphics workstations (SGI,Inc.). Resulting conformers and amino acid side chains of target Alsdomains were refined by molecular dynamics, and strain energies wereminimized using the AMBER95 force field method (Duan et al., J. Comput.Chem. 24:1999-2012 (2003)) and the Powell minimizer (Powell et al.,Math. Program 12:241-254 (1977)).

These approaches optimize side chain interactions where positions of thepeptide backbone atoms are fixed. Preferred conformations weredetermined from extended molecular dynamics in aqueous solvent. Next,the torsion angles of all peptide bonds were adjusted to 180±15°, withminimal constraints. In some cases, molecular dynamics were executed,either with no constraints or with α-helical regions constrained byapplying a 0.4-kJ penalty to the canonical Ramachandran ø and ψ angles.Final global energy minimizations were performed for each model afterthe removal of all constraints and aggregates. Resulting Als N-terminaldomain models were prioritized based on three criteria: (i) mostfavorable strain energy (molecular mechanics); (ii) empirical positionalenergy functions; and (iii) preservation of the spatial arrangement ofpotential disulfide bridging (Godzik et al., J. Mol. Biol. 227:227-238(1992), Bowie et al., Science 253:164-170 (1991), Eisenberg et al.,Methods Enzymol. 277:396-404 (1997), Fischer et al., FASEB J. 10:126-136(1996), Luthy et al., Nature 356:83-85 (1992)). Als models were assessedfor validity in relationship to homology templates using standardmeasures (e-values (Welch et al., Biochemistry 35:7165-7173 (1996),Welch et al., Biochemistry 33:6074-6085 (1994)). Finally, thephysicochemical properties of the Als models were visualized by MOLCAD(Heiden et al., J. Comput. Chem. 14:246-250 (1993)), as implemented inSYBYL and HINT platforms (Kellog et al., J. Comput. Aided Mol. Des.5:545-552 (1991)), such that the physical properties were projected ontothe water-accessible surface of the Als N-terminal domains.

These models indicate that the N-terminal domains of all Als proteinscontain multiple anti-parallel β-sheet domains, consistent with membersof the immunoglobulin superfamily. The results are summarized below inTable III. These proteins typically consist of complex seven-strandedanti-parallel β-sheet domains, from which project loop/coil structures.The β-sheet domains are separated from one another by interposingregions. This structure is often referred to as a beads-on-a-stringmotif. Particularly noted is that virtually all of the Als proteinsmodeled to known adhesin or invasin homologs (Table III). Differentpatterns of similarity were observed among the Als proteins analyzed.For example, all Als proteins examined, except Als7p, shared significanthomology with collagen-binding protein of Staphylococcus aureus.However, the specific primary, secondary, and tertiary homologs variedfor most family members (Table III). For example, Als2p and Als9p sharedan identical primary, secondary, and tertiary homolog.

TABLE III Comparison of homologs among Als proteins Protein Homolog 1Homolog 2 Homolog 3 Als1p Invasin/integrin- Collagen-binding Clumpingfactor binding protein protein Staphylococcus S. aureus (PDB 1n67A)^(b)Yersinia aureus (PDB 1d2p)^(a) pseuodtuberculosis (PDB lcwv)^(a) Als2pCollagen-binding Invasin/integrin- Surface layer protein protein S.aureus binding protein Y. Methanosarcina mazei (PDB ld2p)^(a)pseuodtuberculosis (PDB 1LOQA)^(c) (PDB 1cwv)^(b) Als3p Collagen-bindingInvasin/integrin- Clumping factor protein S. aureus binding protein Y.S. aureus (PDB 1n67A)^(c) (PDB ld2p)^(a) pseuodtuberculosis (PDB1cwv)^(b) Als4p Collagen-binding Invasin/integrin- NS protein S. aureusbinding protein Y. (PDB ld2p)^(a) pseuodtuberculosis (PDB 1cwv)^(b)Als5p Invasin/integrin- Surface layer protein Collagen-binding bindingprotein M. mazei protein S. aureus Yersinia (PDB 1LOQA)^(b) (PDB1d2p)^(c) pseuodtuberculosis (PDB lcwv)^(b) Als6p Collagen-bindingInvasin/integrin- Neuraminidase protein S. aureus binding protein Y.Influenza virus type B (PDB ld2p)^(b) pseuodtuberculosis (PDB lnsca)^(c)(PDB 1cwv)^(b) Als7p Surface layer protein NS NS M. mazei (PDB1LOQA)^(b) Als9p Collagen-binding Invasin/integrin- Surface layerprotein protein S. aureus binding protein Y. M. mazei (PDB ld2p)^(a)pseuodtuberculosis (PDB 1LOQA)^(c) (PDB 1cwv)^(b) Homologes of each Alsprotein were identified by the knowledge-based algorithm described andwere ranked in descending order of structural correlation from 1 to 3.NS, no significant model identified for homology modeling (correlationcoefficient ((r²) ≦70%. PDB, Protein Data Bank code per the NationalCenter for Biotechnology Information format.

Als proteins were also determined to contain N-terminal hypervariableregions that map to predicted loop/coil structures. In this regard,despite the observed differences in substrate-specific adherencemediated by individual Als proteins, large regions of sequence in theN-terminal domains are conserved across this family. However, sevenregions of significant divergence among Als proteins designatedhypervariable regions (HVRs) 1-7, were found. These regions (composed of8 or more amino acids) contained no apparent consensus identity acrossAls proteins and less than 50% consensus conservation. In contrast, theintervening conserved regions (CRs) 1-7, displayed more than 30%consensus identity and more than 50% consensus conservation across Alsproteins. An identity plot and schematic alignment of these amino acidsequences comprising the N-terminal domains (residues 1-420) of Alsproteins with known function is presented in FIG. 11, A and B. Inparticular, homology modeling revealed that the HVRs of different Alsproteins, while distinguishable in sequence, are predicted to conform tosimilar loop/coil structures that project from the β-sheet components ofthe CRs. Thus, the presence of these conserved HVRs indicate that theyare available to interact with host constituents.

In addition to the homology modeling and related determinationsdescribed above, empirical determinations additionally confirm thepredicted structure of the N-terminal domain of Als1p. To test thehypotheses generated by our homology modeling, the structural featuresof the N-terminal domain of Als1p was determined using the complementaryapproaches of CD and FTIR spectrometry. This protein, encompassing aminoacids 17-432 of Als1p, was produced in S. cerevisiae and has beendescribed previously by Fu, et al., Molecular Microbiology, 44:61-72(2002).

Briefly, circular dichroic spectra were recorded with an AVIV 62DSspectropolarimeter (Aviv Biomedical Inc.) fitted with a thermoelectrictemperature controller. Aqueous solutions of Als1p (10 μM inphosphate-buffered saline) were scanned using 0.1-mm light pathdemountable quartz cells at a rate of 10 nm/min from 260 to 185 nm and asample interval of 0.2 nm. Spectra from buffer lacking peptide weresubtracted from sample solutions to minimize light scattering artifacts,and final spectra were an average of 8 scans recorded at 25° C. Theinstrument was routinely calibrated with (+)-10-camphorsulfonic acid (1mg/ml in a 1-mm path length cell) (Johnson et al., Proteins 7:205-214(1990)), and ellipticity was expressed as the mean residue ellipticity(1)MRE (degrees-cm2 dmol-1). The protein concentration was determined byabsorbance at 280 nm based on aromatic amino acid composition of theexpressed Als1p domain (Pace et al, Protein Sci 4:2411-2423 (1995)). TheCD spectra were deconvoluted into helix, β-sheet, turn, and disorderedstructures using Selcon (Sreerama et al, Protein Sci. 8:370-380 (1999))through the internet-based Dichroweb (Lobley et al., Bioinformatics18:211-212 (2002)) interface (cryst.bbk.ac.uk/cdweb/html/home.html).

Infrared spectra of Als1p self-films were recorded at 25° C. on a BrukerVector 22 FTIR spectrometer (Bruker Optics) fitted with a deuteratedtriglycine sulfate detector at a gain of 4, averaged over 256 scans, andat a resolution of 2 cm-1. Fifty micrograms of the protein in 50 μl ofphosphate-buffered saline were spread onto the surface of a 50×20×2-mmgermanium attenuated total reflectance sample crystal (PikeTechnologies) and allowed to dry. The dry protein self-film was thenhydrated with D2O for 1 h prior to recording the infrared spectra. AmideI bands of the infrared spectra were analyzed for secondaryconformations by area calculations of component peaks with curve-fittingsoftware (GRAMS/32, Version 5; Galactic). The frequency limits for thevarious conformations were as follows: α-helix (1662-1645 cm-1), β-sheet(1637-1613 and 1710-1682 cm-1), β-turn loops (1682-1662 cm-1), anddisordered structures (1645-1637 cm-1) (50-52).

Circular dichroism results of the N-terminal domain of Als1p are shownin FIG. 12A and reveal a dichroic minimum at 217 nm and strong positivedichroic maximum near 200 nm. These features are characteristic of aprotein having a dominant anti-parallel β sheet component. Deconvolutionof the CD spectra indicated that the protein assumed conformations of50.1% β sheet, whereas other structure class contributions includedisordered structures (26.9%), turn structures (19.3%), and α-helix(3.7%).

As shown in FIG. 12B, FTIR measurements of a self-film of the hydratedAls1p strongly corroborated that the sample has a dominant β-sheetconformation. These spectra revealed strong low frequency amide I bandswith peaks centered at 1634 and 1628 cm-1 and a weak high frequency bandcentered at 1685 cm-1. This frequency splitting of the protein amide Iinfrared spectra into high and low frequency components has been shownto be typical of the effect of transition dipole coupling betweenintermolecular anti-parallel β-sheets (Halverson et al., J. Am. Chem.Soc. 113:6701-6703 (1991)). Curve fitting of the spectra indicated thatthe protein construct is ˜57.2% antiparallel β-sheet. Other secondarystructural conformations from curve fitting of the IR spectra includedisordered structures (20.5%), turn components (13.3%), and α-helix(9.0%).

Taken together, the FTIR and CD data further corroborate that the Nterminus of Als1p contains predominant domains of anti-parallel β-sheetstructure containing minor α-helical and turn components, interposed byless structured regions.

Three-dimensional models further indicate Physicochemical distinctionsamong Als N-terminal domains. In this regard, molecular models indicateddifferences in predicted physicochemical attributes of the N-terminaldomains of Als proteins that likely influence their interactions withhost cells and several substrates. As shown in FIG. 13, Als proteins areseparable into three distinct groups based on surface distributions ofhydrophobicity, charge, and hydrogen bonding potential. Als1p, Als3p,and Als5p each share similar patterns of these properties and thus areconsidered the Als group A. In contrast, the predicted physicochemicalproperties of Als6p and Als7p N-terminal domains (Als group B) havestriking differences from those of the Als group A (FIG. 13). Whereasthe cationic potential in Als group A members is typically segregatedfrom their neutral or anionic facets, positive charge is broadlydistributed across the entire surface of the Als group B members Als6pand Als7p. Finally, the N termini of Als2p, Als4p, and Als9p appear toconstitute a third group of Als proteins (the Als group C) that differstructurally from either the Als group A or B proteins. The Als group Cproteins would appear to be more similar to the Als group A than Alsgroup B proteins in terms of hydrophobic or electrostatic distribution.

Several proteins with adhesive function have been identified in C.albicans. Hwplp has been shown to mediate adherence to buccal epithelialcells by acting as a substrate for mammalian transglutaminase (5). EAP1was recently identified by heterologous expression in S. cerevisiae andmediates adherence to polystyrene and renal epithelial cells in vitro(7). Of the eight members of the Als protein family, only Als1p andAls5p have been studied from a functional perspective. Heterologousexpression of Als1p has been shown to mediate binding to human vascularendothelial cells and epithelial cells, a finding that has beenconfirmed in C. albicans through gene disruption studies (Fu et al.,Mol. Microbiol. 44:61-72 (2002), Fu et al., Infect. Immune. 66:1783-1786(1998)). Heterologous expression of ALS5 in S. cerevisiae confersadherence to collagen, fibronectin, bovine serum albumin, and laminin(Gaur et al., Infect. Immune. 65:5289-5297 (1997), Gaur et al., Infect.Immun. 67:6040-6047 (1999), Gaur et al., Cell Commun. Adhes. 9:45-57(2002)). No large scale comparison of the substrate specificities of C.albicans adhesins has been performed. In this study, we compared theadhesive properties of a structurally diverse group of Als proteinfamily members. Our data demonstrate that the Als proteins comprise adiverse family of surface proteins with an overlapping spectrum ofspecificities for adherence to a variety of human substrates (FIG. 8).Further, results from the present domain exchange experiments indicatethat the N-terminal domains of Als proteins confer the specificity oftheir substrate adherence profiles.

In addition to mediating adherence, our data suggest that Als proteinsalso can function as invasins. Interestingly, whereas both Als1p andAls3p expressing S. cerevisiae demonstrated similar endothelial celladherence, Als3p-expressing S. cerevisiae underwent internalization at amuch higher rate. These results indicate that endocytosis is not simplyan extension of adherence but rather a distinct process that can beinfluenced by the ligand-receptor interaction. It is likely thatdifferences in N-terminal sequences in Als proteins mediate thesedistinct functions, as is the case with adherence.

The physicochemical properties of protein domains as distributed inthree-dimensional space are crucial structural features governingreceptor-ligand interactions (Eisenberg et al, J. Mol. Biol. 179:125-142(1984), Waring et al., Protein Peptidew Lett. 3:177-184 (1996), Hancocket al., Lancet 349:418-422 (1997)). The Als proteins shareconformational features characteristic of other adhesins and invasins ofthe immunoglobulin superfamily. However, individual Als proteinsdiffered in their primary homolog, a finding consistent with theexperimental data indicating that members of the Als family exhibitdifferent substrate-binding profiles. Collectively, these patterns ofAls homologies indicate that, whereas Als protein members share a globalsimilarity in structure and predicted fold, there exists structuraldifferences among distinct Als proteins that are responsible for theirdifferences in function.

The results described above relating to the Als family member structuraldeterminations corroborate the homology modeling, which indicates thatthe N-terminal regions of Als1p are composed predominantly ofanti-parallel β-sheet domains containing loop/coil structures, withlesser amounts of relatively unstructured regions. These features areindicative motifs of members of the immunoglobulin superfamily. Theseresults show significant predictive correlation with circular dichroismstudies of Als5p (Hoyer et al., Yeast 18:49-60 (2001)), indicating thatthe N-terminal domain of Als5p is characterized by a relativepredominance of anti-parallel β-sheet and loop/coil regions. Thus, it ishighly likely that all members of the Als protein family exhibit thisoverall structure. In particular, the structural results above are alsoconsistent with the homology models that indicate that many of the HVRscorrespond to the flexible loop/coil structures projecting from β-sheetdomains in the N termini of distinct Als proteins. Collectively, theseresults indicate that these structures are integral tosubstrate-specific binding by Als proteins (FIG. 14). Consistent withthe results above, analogous regions of mannose-binding lectin,α-agglutinin, and other members of the immunoglobulin superfamily appearto confer substrate binding specificity (Zhao et al., Hybrid Hybridomics21:25-36 (2002), Wojciechowicz et al., Mol. Cell. Biol. 13:2554-2563(1993)). Furthermore, mutations of these variable loop regionssignificantly alter substrate binding in these homologous proteins (Renzet al., J. Cell Biol. 125-1395-1406 (1994), Viney et al., J. Immunol.157:2488-2497 (1996)).

The three-dimensional modeling results further indicate that N-terminaldomains of individual Als proteins possess distinctive molecularsignatures that relate to their adhesive profiles. These signaturesincorporate parameters such as surface area, hydrophobicity, andelectrostatic charge, yielding configurations that distinguishstructural relationships among Als proteins. For example, Als proteinsthat bind to multiple substrates, such as the Als group A members(Als1p, Als3p, and Als5p), have similar predicted N-terminal profiles interms of steric bulk, hydrophobic distribution, and electrostaticpotential. Yet, even within this group, specific physicochemicaldistinctions exist that can govern functional differences within thegroup (FIG. 13). In contrast, Als proteins with reduced adhesivecapacity have surface features predicted to be distinct from the Alsgroup A proteins in multiple physicochemical properties, includinghydrophobicity and electrostatic potential. It is highly likely that theaggregate effects of differences in these structural features confer thespecific functional properties of distinct Als proteins.

Extensive genetic variability has been demonstrated within the ALS genefamily. Sequence variation in specific ALS genes of different isolatesof C. albicans has been observed (Zhang et al., Genome Res. 13:2005-2017(2003), Hoyer et al., Yeast 18:49-60 (2001)), and not all members of theALS family are present in all isolates. Even significant sequencedivergence between two different alleles in a single isolate have beenfound (Zhao et al., Microbiology 149:2947-2960 (2003), Zhang et al.,Genome Res. 13:2005-2017 (2003)). This degree of genetic variabilitywould suggest that these proteins may undergo rearrangement or mutationat a relatively high frequency. Such a mechanism would provide theorganism with the ability to generate the high degree of structural andfunctional diversity demonstrated in this study. Indirect support forthis hypothesis is provided by a recent study of allelic variation ofALS7, which suggested both that this gene is both hypermutable and thatthese mutations are subject to selective pressure (Zhang et al., GenomeRes. 13:2005-2017 (2003)).

Collectively, the above results indicate an analogy between antibodiesand Als proteins at both the structural and functional level. Forexample, the homology modeling underscores the similarities instructural configurations of these families, with hypervariabilitytargeted to localized domains within an otherwise stable framework (e.g.HVRs of Als proteins and Fab regions in immunoglobulins). Further, aswith antibodies, the genetic variability of the ALS gene family mayprovide the opportunity for Candida to display a diverse array ofproteins with a spectrum of specificity in adherence and invasion. Theavailability of such a group of related proteins is likely to improvethe ability of the organism to colonize and invade different anatomicaland physiological niches during infection.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

Example VII Vaccination with Rals1p-N Improves Survival During MurineDisseminated Candidiasis by Enhancing Cell-Mediated, not Humoral,Immunity

This example shows that immunizing BALB/c mice with the recombinantN-terminus of Als1p (rAls1p-N) improved survival during subsequentchallenge with a lethal inoculum of C.

albicans. The protective 20 μg dose of rAls1p-N significantly increasedCandida-stimulation of Th1 splenocytes and increased in vivo delayedtype hypersensitivity. In contrast, antibody titers did not correlatewith protection. Finally, the vaccine was not protective in Tcell-deficient mice but was protective in B cell-deficient mice. Thesedata indicate that the mechanism of action of the rAls1p-N vaccine isstimulation of cell mediated, rather than humoral, immunity against C.albicans.

The C. albicans used in the study was SC5314, a well-characterizedclinical isolate that is highly virulent in animal models (Spellberg etal., Infect Immun. 71:5756-5764 (2003)) was supplied by W. Fonzi(Georgetown Univeristy). The organism was serially passaged three timesin yeast peptone dextrose broth (Difco) prior to infection.

The mice strains used in the study were female BALB/c mice obtained fromthe National Cancer Institute (Bethesda, Md.). To explore the impact ofage on vaccine efficacy, both juvenile mice (8-10 weeks) and retiredbreeders 6 months) were utilized. Female B cell-deficient mice bearing ahomozygous deletion of the igh loci (C.129B6-IgH-JhdtmlDhu), Tcell-deficient nude mice (C.Cg/AnBomTac-FoxnlnuN20), and congenicwild-type BALB/c littermates were obtained from Taconic Farms(Germantown, N.Y.). Mice were housed in filtered cages with irradiatedfood and autoclaved water ad libitum. For survival experiments, micewere immunized with varying doses of antigen (see below) andsubsequently infected via the tail vein with the appropriate inoculum ofC. albicans SC5314 blastospores, or PBS (Irvine Scientific, Irvine,Calif.) control. Results of replicate survival studies were combined ifthe individual datasets demonstrated no statistical heterogeneity (seebelow). All procedures involving mice were approved by the institutionalanimal use and care committee, following the National Institutes ofHealth guidelines for animal housing and care.

The rAls1p-N immunization procedures described below were performed asfollows. Briefly, rAls1p-N (amino acids 17 to 432 of Als1p) was producedin S. cerevisiae and purified by gel filtration and Ni-NTA matrixaffinity purification (Fu et al., Molec. Microbiol. 44:61-72 (2002)).The amount of protein was quantified by modified Lowry assay. A highdegree of purity (≈90%) was confirmed by SDS-polyacrylamide gelelectrophoresis as well as circular dichroism and FTIR, as describedabove. Mice were immunized by intraperitoneal (ip) injection of rAls1p-Nmixed with complete Freund's adjuvant (CFA, Sigma-Aldrich) at day 0,boosted with another dose of the antigen with incomplete Freund'sadjuvant (IFA, Sigma-Aldrich) at day 21, and infected two weeksfollowing the boost.

Resultant Antibody titers were determined by ELISA in 96 well plates.Briefly, wells were coated with 100 μl per well of 5 μg/ml rAls1p-N inPBS. Mouse sera were incubated for 1 h at room temperature following ablocking step with tris buffer saline (TBS) (0.01 M TrisHCl, pH 7.4,0.15 M NaCl) containing 3% bovine serum albumin. The wells were washed 3times with TBS containing 0.05% Tween 20, followed by another 3 washeswith TBS. Goat anti-mouse secondary antibody conjugated with horseradishperoxidase (Sigma) was added at a final dilution of 1:5000 and the platewas further incubated for 1 h at room temperature. Wells were washedwith TBS and incubated with substrate containing 0.1 M citrate buffer(pH 5.0), 50 mg/ml of o-phenylenediamine (Sigma), and 10 μA of 30% H₂O₂.The color was allowed to develop for 30 min after which the reaction wasterminated by adding 10% H₂SO₄ and the optical density (OD) wasdetermined at 490 nm in a microtiter plate reader. Negative controlwells received only diluent, and background absorbance was subtractedfrom the test wells to obtain final OD readings. The ELISA titer wastaken as the reciprocal of the last serum dilution that gave a positiveOD reading (i.e. >mean OD of negative control samples+2standarddeviation).

Other methods described below were performed as follows. Briefly, C.albicans-induced cytokine profiles were performed to determine theeffect of the rAls1p-N vaccine on cell-mediated immunity and in vivocytokine profiles. Mice were immunized as described above. Two weeksafter the final boost, splenocytes were harvested and cultured incomplete media at a density of 4×10⁶ cells/ml as previously described(Spellberg et al., Infect. Immun. 71:5756-5764 (2003)). To stimulatecytokine production, splenocytes were co-cultured with heat-killed C.albicans SC5314 germ tubes. We used heat-killed C. albicans in lieu ofrAls1p-N to stimulate the splenocytes to mimic the in vivo situationduring infection. The C. albicans cells were pre-germinated in RPMI-1640with glutamine (Gibco BRL) for 90 minutes to induce expression of Als1p(Fu et al., Molec. Microbiol. 44:61-72 (2002)). The resulting C.albicans germ tubes were heat-killed by incubation for 90 minutes at 60°C. (Ibrahim et al., Infect. Immun. 63:4368-74 (1995)). The heat-killedfungi were added to the splenocyte cultures at a density of 2×10⁷pseudohyphae/ml (ratio of five fungi to one leukocyte). After 48 h,splenocytes were profiled for Th1 (CD4+IFN-□+IL-4−), Th2(CD4+IFN-□-IL-4+), or CD4+IL-10+ frequencies by intracellular cytokinedetection and flow cytometry, as previously described (Spellberg et al.,Infect. Immun. 71:5756-5764 (2003)). Three-color flow cytometry wasperformed on a Becton-Dickinson FACScan instrument calibrated withCaliBRITE beads (Becton Dickinson, San Jose, Calif.) using FACSCompsoftware as per the manufacturer's recommendations. During dataacquisition, CD4+ lymphocytes were gated by concatenation of forward andside scatter, and FITC-anti-CD4 antibody fluorescence properties. Datafor each sample were acquired until 10,000 CD4+ lymphocytes wereanalyzed. Results are presented as the median±25th and 75th quartiles ofthe percentage of all gated lymphocytes that were Th1 or Th2 cells.

Footpad swelling was determined by the method of Oomura et al (41).Briefly, mice were immunized with the appropriate dose of rAls1p-N orCFA alone as described above. Two weeks following the boost, baselinefootpad sizes of immunized mice were measured using an electronicdigital caliper. Fifty μg of rAls1p-N in 25 μl of PBS was injected intothe right footpads, and PBS alone injected into the left footpads of theimmunized mice. Twenty-four hours later the footpads were againmeasured. Antigen-specific footpad swelling was calculated as: (rightfootpad thickness at 24 h−right footpad thickness at baseline)−(leftfootpad thickness at 24 h−left footpad thickness at baseline).

The non-parametric Log Rank test was utilized to determine differencesin survival times of the mice. Titers of antibody, frequency of Th1 orTh2 lymphocytes, and footpad swelling were compared by the Steel testfor non-parametric multiple comparisons (Rhyne et al., Biometrics23:539-49 (1967) or the Mann Whitney U test for unpaired comparisons, asappropriate. Correlations were calculated with the Spearman Rank sumtest. To determine if heterogeneity existed in replicate survivalstudies, the Kolmogorov-Smirnov test was utilized. P values<0.05 wereconsidered significant.

To determine the most effective dose of the rAls1p-N immunogen, a10⁷-fold dose range was evaluated (20 pg to 200 μg per mouse). Femaleretired breeder BALB/c mice were immunized with rAls1p-N plus adjuvant(CFA/IFA) or adjuvant alone. Immunized mice were bled 2 weeks afterboosting to determine anti-rAls1p-N antibody titers (see below). Themice were subsequently infected with a lethal inoculum of C. albicans(2×10⁵ blastospores). The survival data from repeat experiments werecombined since the individual experiments demonstrated no statisticalheterogeneity (p>0.05 by Kolmogorov-Smirnov test). The 20 μg dose ofrAls1p-N resulted in long-term survival of 25% of the infected mice, anda significant increase in overall survival compared to adjuvant alone(p=0.044 by Log Rank test, FIG. 1). Neither 10-fold higher (FIG. 15) norlower (data not shown) doses significantly increased survival comparedto adjuvant alone. These results indicate that an intermediate dose ofthe rAls1p-N vaccine induces protection against murine disseminatedcandidiasis.

The above findings established a protective dose for the rAls1p-Nvaccine. Next the efficacy of the vaccine was evaluated in a morerapidly lethal model of mice infected with 10⁶ blastospores (mediansurvival 3 vs. 11 days for 10⁶ vs. 2×10⁵ inocula in unvaccinated mice,respectively). Again the data from repeat studies were combined as theresults of the individual experiments demonstrated no statisticalheterogeneity (p>0.05 by Kolmogorov-Smirnov test). When administered asa 20 μg dose+CFA to Balb/c mice infected with 10⁶ C. albicansblastospores, the rAls1p-N vaccine more than doubled the median survivaland resulted in a significant increase in overall survival versusunvaccinated controls (p=0.001 by Log Rank test, FIG. 16A). To determineif the age of the mice influenced their response to the rAls1p-Nvaccine, we tested it in juvenile mice. A similar survival benefit wasfound when juvenile mice were vaccinated and infected with the same highinoculum (p=0.02 by Log Rank test, FIG. 16B).

Although the 200 μg dose of rAls1p-N resulted in inferior protectioncompared to the μg dose, only the 200 μg dose of antigen induced asignificant increase in serum anti-Als1p antibody titers (p≦0.005 for200 μg dose vs. all other groups, FIG. 17). No significant increases inanti-Als1p antibody titers were detected at the intermediate, protectiveantigen dose (p=0.1 for 20 μg vs. adjuvant). When the serum anti-Als1pantibody titers of individual mice were plotted against the survivaltime of each mouse, no correlation between antibody titer and survivalwas found (R²=0.03, p>0.05 by the Spearman rank sum test). Indeed, miceimmunized with the highest dose of antigen (200 μg) had anti-rAls1p-Nantibody titers in excess of 1:100,000, but had survival durations nodifferent from mice immunized with lower doses of antigen whose titerswere at the lower limit of detection (˜1:100). These results indicatethat protection induced by the rAls1p-N vaccine does not appear tocorrelate with antibody titers.

Since humoral immunity did not correlate with rAls1p-N-inducedprotection, we examined the cell-mediated immune response induced byprotective and non-protective doses of rAls1p-N. Mice were immunizedwith 0.2, 20, or 200 μg of rAls1p-N, or adjuvant alone, as above. Twoweeks after the boost, splenocytes were harvested and cultured in thepresence of heat-killed, pre-germinated C. albicans, which are known toexpress Als1p (Fu et al., Molec. Microbiol. 44:61-72 (2002)). Following48 h of culture, splenocytes were harvested for intracellular cytokinedetection by flow cytometry. Only the lymphocytes from mice immunizedwith the protective 20 μg dose of antigen developed a significantlyincreased frequency of Th1 cells compared to mice given adjuvant alone(p=0.03, FIG. 18). No significant differences in Th2 frequency (FIG. 18)or in the frequency of IL-10⁺ T lymphocytes (data not shown) weredetected between mice immunized with adjuvant or any of the doses ofantigen.

To confirm that Type 1 immunity was stimulated by r-Als1p-N in vivo,delayed type hypersensitivity was tested by footpad swelling. Only micevaccinated with the protective 20 μg dose of rAls1p-N developed asignificantly increased delayed type hypersensitivity reaction comparedto control, and this response was also significantly greater than thatinduced by the non-protective 0.2 and 200 μg doses (FIG. 19, p<0.05 forall comparisons versus 20 μg dose, by the non-parametric Steel test).Collectively, these results indicate that a protective dose of therAls1p-N antigen induced significant Th1 polarization and delayed typehypersensitivity reaction.

To define the role of antibody and T-cells in vaccine-mediatedprotection, B cell-deficient, T-cell deficient nude, or congenic BALB/cwild-type control-mice were immunized with 20 μg of rAls1p-N plusadjuvant or adjuvant alone, and infected with a lethal inoculum (8×10⁵blastospores) of C. albicans. B cell-deficient mice trended to beingmore resistant to infection, whereas T cell-deficient mice were moresusceptible, than were wild-type control mice given adjuvant alone(p=0.065 and 0.01 for B cell-deficient and T cell-deficient mice versuswild-type adjuvant-treated, respectively, FIG. 20). Finally, therAls1p-N vaccine maintained its efficacy in B cell-deficient mice(p=0.04 for rAls1p-N vaccinated versus adjuvant alone, FIG. 6) but wasineffective in T cell-deficient mice (p=0.4 for rAls1p-N vaccinatedversus adjuvant alone, FIG. 20). These results indicate that the Als1pvaccine is effective in B cell-deficient mice but not in T-celldeficient nude mice.

Described above are the results showing that immunization with theN-terminus of this protein improved survival of both juvenile and matureBALB/c mice during subsequent hematogenously disseminated candidiasis.In particular, an intermediate dose of rAls1p-N (20 μg) providedsuperior protection compared to both lower doses and a higher dose (200μg). Nevertheless, the non-protective 200 μg dose of rAls1p-N wasimmunogenic, as it induced 100-fold higher titers of antibody than didthe protective 20 μg dose.

The inverted U-shaped dose-response efficacy curve, with lowerprotection at the highest dose of rAls1p-N, is reminiscent of theclassical studies of Parish et al., who first described the inverserelationship between the induction of humoral and cell-mediated immunityby a given dose of antigen. In the context of Parish's seminal data, aninverted U-shaped dose-response efficacy curve could be explained if: 1)vaccine efficacy depended on cell-mediated immunity and, 2) intermediatedoses of rAls1p-N stimulated superior cell-mediated immunity compared tothe high, antibody-stimulating dose. We therefore hypothesized that theinverted U-shaped dose response efficacy curve seen with the rAls1p-Nvaccine was due to superior induction of cell-mediated immunity by theprotective, intermediate doses of antigen.

To test this hypothesis, the ability of high, intermediate, and lowdoses of antigen to stimulate Th1 cells and delayed-typehypersensitivity were determined. To stimulate cytokine-production fromsplenocytes, we specifically activated the cells by exposure toheat-killed C. albicans, instead of rAls1p-N, to mimic the in vivosituation during infection. Only the protective 20 μg dose,significantly increased the frequency of C. albicans-stimulated, splenicTh1 lymphocytes. The frequency of Th1 cells seen in ex vivo C.albicans-stimulated splenocytes was similar to that detected in vivoduring disseminated candidiasis in mice (59), underscoring the relevanceof this approach.

To determine if the detected ex vivo Th1 cells were of functionalsignificance in vivo, we compared the delayed type hypersensitivityinduced by different doses of rAls1p-N immunization. Concordant with thefrequency of Th1 cells, only the protective 20 μg dose of rAls1p-Nstimulated a significant in vivo delayed type hypersensitivity reaction.These results are consistent with the hypothesis that vaccine-inducedprotection was due to induction of Type 1, cell mediated immunity.Surprisingly, despite induction of markedly elevated antibody titers bythe 200 μg dose of rAls1p-N, we did not find an increase in splenic Th2lymphocytes in mice vaccinated with this dose. One possible explanationis that Th2 cells were activated in peripheral lymph nodes rather thanthe spleen. Alternatively, other T cell populations (e.g. NKT cells) mayhave been responsible for inducing the high antibody titers seen inresponse to the 200 μg dose of rAls1p-N.

The lack of correlation between antibody titer and protection did notcompletely exclude a role of antibodies in mediating vaccine-inducedprotection. For example, ELISA titers are the result of enumeration ofantibodies with a variety of specificities and affinities. Therefore,the possibility that small subsets of antibodies were generated that didparticipate in vaccine-mediated protection could not be excluded bymeasuring antibody titer. To confirm the role of cell-mediated and nothumoral immunity in rAls1p-N vaccine-mediated protection, we tested theefficacy of the vaccine in B cell- and T cell-deficient mice. Bcell-deficient mice trended to being more resistant to disseminatedcandidiasis than wild-type controls, and the efficacy of the vaccine wasnot abrogated in B cell-deficient mice. In contrast, T cell-deficientmice were more susceptible to disseminated candidiasis than werewild-type controls, and the efficacy of the vaccine was lost in Tcell-deficient mice. Our findings therefore confirm that the efficacy ofthe rAls1p-N vaccine is dependent of induction of T-cell mediated, andnot primarily humoral, immunity. As well, because B cell-deficient micewere not more susceptible to disseminated candidiasis than congenic wildtype littermates, antibody is not a dominant effector againstdisseminated candidiasis in this model.

In sum, we report that the novel rAls1p-N vaccine mediates protectionagainst experimental disseminated candidiasis by inducing cell-mediatedrather than humoral immunity. Enhancement of the modest protectiveeffect of the rAls1p-N vaccine may therefore be accomplished withadditional priming of cell-mediated immunity using optimized adjuvantsand/or cytokines, or an alternate route of immunization. Indeed, in ourongoing studies we have already found a marked increase in efficacy byadministering rAls1p-N subcutaneously as compared to intraperitoneally.

Example VIII The Anti-Candida albicans rAls1p-N Vaccine Reduces FungalBurden and Improves Survival in Both Immunocompetent andImmunocompromised Mice

This example describes enhancement of the efficacy of the rAls1p-Nvaccine described in example VII when administered by a subcutaneous(SQ) route in both immunocompetent and immunocompromised mice.Initially, the efficacy of the rAls1p-N vaccine in immunocompetent mice.rAls1p-N, encompassing amino acids 19-433 of the full length protein,was produced in S. cerevisiae and purified as described above. Controlpreparation was similarly purified from S. cerevisiae transformed withan empty plasmid. BALB/c retired breeder mice (25-30 g) were immunizedby SQ injection of rAls1p-N (20 μg) or control preparation mixed withComplete Freund's Adjuvant (CFA) at day 0, followed by a booster dose inIncomplete Freund's Adjuvant (IFA) at day 21. Two weeks following theboost, the immunogenicity of the vaccine was confirmed by evaluating theintensity of the footpad swelling reaction as a marker of delayed typehypersensitivity (DTH), as previously described. Vaccinated mice hadmarked increases in rAls1p-N specific DTH (FIG. 21).

The efficacy of the rAls1p-N vaccine was evaluated by determining theimpact of rAls1p-N vaccination on survival in infected BALB/c mice (FIG.22A). Vaccinated or control mice were infected via the tail-vein withrapidly lethal inocula (2.5−5×10⁵ blastospores) of C. albicans. We havepreviously shown that mice infected such inocula die of overwhelmingseptic shock (Spellberg et al., J. Infect. Dis. In press (2005)).Vaccination markedly prolonged time to death (p<0.05 for both inocula byLog Rank test) and improved 30 day survival (50-57% vs. 0%, p<0.05 forboth inocula by Fisher's Exact test).

The impact of vaccination on tissue fungal burden during hematogenouslydisseminated candidiasis was then determined. Fourteen days followingthe boost, vaccinated and control BALB/c mice were infected with via thetail-vein with 5×10⁵ blastospores of C. albicans SC5314. Six daysfollowing infection, prior to onset of the first deaths in the controlarm, kidneys were harvested, homogenized, and quantitatively cultured inSabouraud dextrose agar (Difco) (18). SQ vaccination with rAls1p-Nresulted in a median 1.5 log CFU/g decrease in kidney fungal burdencompared to control (p=0.01 by Wilcoxon Rank Sum test, FIG. 22B).

The efficacy of the rAls1p-N vaccine also was assessed inimmunocompromised mice. Having demonstrated efficacy in immunocompetentmice, the potential for the rAls1p-N vaccine to induce immunity in andprotect neutropenic mice from disseminated candidiasis also wasevaluated. Vaccinated BALB/c mice were made neutropenic byadministration of cyclophosphamide (200 mg/kg ip on day −2, and 100mg/kg ip on day +9 relative to infection, resulting in approximately 12days of neutropenia, as described (Sheppard et al., Antimicrob. Agents.Chemother. 48:1908-11 (2004)). Footpad swelling reaction was performed 2days after the first dose of cyclophosphamide. Vaccinated neutropenicmice developed DTH reactions of similar magnitude to immunocompetentmice (FIG. 23A vs. 1, experiments performed in parallel). In neutropenicmice infected via the tail-vein with 2.5×10⁴ blastospores of C.albicans, vaccination also resulted in significant improvements in timeto death (p=0.007 by Log Rank test vs. Control), median survival time(>21 vs 12 d, p=0.008 by Wilcoxon Rank Sum Test), and overall survival(88% vs. 38%, p=0.005 by Fisher's Exact test) (FIG. 23B).

To determine the efficacy of rAls1p-N vaccination in mucosal infection,the vaccine was tested in a murine oropharyngeal candidiasis (OPC) model(Kamai et al., Infect. Immun. 70:5256-8 (2002) and Kamai et al.,Antimicrob. Agents Chemother. 45:3195-97 (2001)) Vaccinated mice weretreated with cortisone acetate (225 mg/kg SQ on days −1, 1, and 3relative to infection) and infected sublingually as described. Tongueswere excised on day 5 post-infection. Because colony forming units ofhomogenized tongues cannot distinguish between invasive infection andsurface-adherent colonization, we evaluated extent of invasion byhistopathology. A blinded observer (BJS) scored each section by scanningalong the entire length of the tongues and quantifying the severity offungal lesions per 40× high-powered field (0=no lesion, 1⁺=mild mucosalinflammation, 2⁺=significant inflammation restricted to the epithelium,3⁺=inflammation extending through the entire epithelial layer,4⁺=inflammation extending into the subepithelium). To avoid samplingbias, two sections of each tongue, separated by at least fiveintervening tissue sections, were scored. All control mice developedmarked fungal invasion of their tongues in numerous locations, whileonly two vaccinated mouse developed any tongue lesions. In total, themedian number (75^(th), 25^(th) quartile) of lesions per tongue incontrol mice was 6.5 (8, 5.75) as compared to 1 (2.5, 0) for vaccinatedmice (p=0.03 by Wilcoxon Rank Sum test). Semi-quantitative evaluation ofthe severity of infection demonstrated a significant reduction invaccinated mice compared to controls (FIG. 24, p=0.03 by Wilcoxon RankSum test).

To determine the efficacy of rAls1p-N or rAls3-p-N vaccination inmucosal infection, these two vaccines in a murine model of vaginalcolonization (Clemons et al., Infect. Immun. 72: 4878-80 (2004); Fidel.Int Rev Immunol. 21: 515-48 (2002) and Wozniak et al., Infect Immun. 70:5790-9 (2002)). Vaccinated mice were treated with estrogen (30 μg, givenSQ) on day −3 relative to infection and then challenged in the vaginawith 10 μl phosphate buffered saline containing 10⁶ blastospores of C.albicans. Vaginas were excised on day 3 post-inoculation, homogenizedand serial dilutions were plated on YPD plates. Colony forming units(CFU) were enumerated 24-48 h following incubation of plates at 30-35°C. Vaginas collected from mice vaccinated with rAls3p-N but not thosecollected from mice vaccinated with rAls1p-N had lower CFU than vaginascollected from control mice (i.e mice vaccinated with CFA alone) (FIG.25, p=0.01 by Wilcoxon Rank Sum test).

In light of the increasing incidence of candidemia and its continuinghigh mortality rate, development of a vaccine against Candida spp. is ofgreat importance. The results described above show that SQ vaccinationwith rAls1p-N resulted in marked improvement in survival and significantreductions in fungal burden during otherwise rapidly fatalhematogenously disseminated candidiasis in both immunocompetent andimmunocompromised mice. Of interest are the kidney fungal burden resultsfrom individual vaccinated mice, demonstrating that approximately halfthe mice had kidney fungal burdens under 5 log CFU/g. We have previouslyfound that the threshold of kidney fungal burden indicative of a fatalinfection is 5 log CFU/g; mice with kidney fungal burdens above thislevel typically die from infection, whereas mice with kidney fungalburdens below this burden survive the infection (Spellberg et al., J.Infect. Dis. In press (2005) and (Spellberg et al., Infect. Immun.71:5756-5764 (2003)). Therefore, breakthrough deaths in the vaccinatedgroup likely reflect high fungal burden in spite of vaccination. Themouse to mouse variations in tissue fungal burden may reflect thecomplexities of host-pathogen interactions and/or variable vaccineresponsiveness.

In summary, the rAls1p-N vaccine can be used for the treatment,reduction in severity and/or prevention of increasingly common andhighly lethal disseminated candidiasis. The vaccine is efficacious inimmunocompetent mice, and efficacy is retained even in neutropenic andcorticosteroid-treated hosts. Finally, the vaccine can protect againstmucocutaneous candidiasis including vaginal and oropharyngealcandidiasis

Example IX Effectiveness of Als Vaccines Against S. aureus Infections

This Example shows that Als proteins from C. albicans improves survivalof animal models infected with S. aureus.

Als adhesins of C. albicans were identified to be significantlyhomologous to adhesins on S. aureus. This characteristic was used todesign and implement an effective vaccine against S. aureus using Alsadhesins. Briefly, the C. albicans ALS family is comprised of at least 9genes (Hoyer et al., Genetics 157:1555-67 (2001); Hoyer L L., TrendsMicrobiol. 9:176-80 (2001)). As described previously, Als proteinsfunction as adhesins to biologically relevant substrates (Fu et al.,Molec. Microbiol. 44:61-72 (2002); Gaur and Klotz, Infect. Immun.65:5289-94 (1997); Zhao et al., Microbiology 150:2415-28 (2004); Oh etal., Microbiology 151:673-81 (2005); Zhao et al. Microbiology151:1619-30 (2005)); Hoyer et al., Mol. Microbiol. 15:3954 (1995)). Inparticular, the N-termini of Als1p and Als3p are significantlyhomologous to surface proteins expressed by pathogenic S. aureus,including collagen binding protein and clumping factor (Table IV;Sheppard et al. J. Biol. Chem. 279:30480-89 (2004)).

TABLE IV Homology of Als proteins to various pathogenic adhesins andinvasions Protein Homologue 1 Homologue 2 Als1p Collagen binding proteinof Clumping factor of S. aureus: ≧95% homology S. aureus: ≧90% homologyAls3p Collagen binding protein of Clumping factor of S. aureus: ≧95%homology S. aureus: ≧80% homology Als5p Invasin/integrin-binding Surfacelayer protein M. protein Y. pseuodtuberculosis mazei

The homology calculation provided above in Table IV takes into accountboth features of sequence alignment and 3-dimensional surface structure.Homology of Als1p was calculated to be greater than 95% or 90% comparedto collagen binding protein or clumping factor of S. aureus (r²≧90%;Sheppard et al., supra). Similarly, homology of Als3p was calculated tobe greater than 95% or 80% compared to collagen binding protein orclumping factor of S. aureus (r²≧90%).

To corroborate the above findings, homology and threading methods wereemployed to model structure-function congruence between Als1p and S.aureus clumping factor A (ClfA-PDB code: cln67A). These methods assessedspecific homologies in primary structure, 3-D conformation and patternanalyses were conducted to seek analogous functional motifs. Forexample, BLASTP, PROSITE and JALVIEW methods were employed to analyzesimilarities and differences in ALS versus ClfA primary sequences (Yountet al. Antimicrob. Agents Chemother. 48:4395-4404 (2004) and Yount andYeaman. Proc. Natl. Acad. Sci. USA 101:7363-7368 (2004)). Internet-basedapplications including 3-D PSSM were then used to prioritize potentialALS homologues for further analysis (Sheppard, et al. J. Biol. Chem.279:30480-30489 (2004)). Along with resulting data, the PHYREapplication (Kelley, L., R. Bennett-Lovsey, A. Herbert, and K. Fleming;website is as follows: http://www.sbg.bio.ic.ac.uk/˜phyre/) was used toconduct topology mapping and to identify 3-dimensional motifs shared byproteins with greatest structural or functional homology to selected ALSproteins for the purpose of identifying putative shared functionalmotifs. The above methods are widely available in public domain and usedin a variety of proteomic and structural biology applications. Based onthe above homology and threading method results a consensus offunctional site homologies between Als1p and ClfA was generated andmapped to specific residues of the Als1p model constructed on ClfA.Several particular findings emanated from these modeling analyses as setforth below.

First, significant homology was identified between the N-terminalregions of Asl1p and ClfA in secondary structure and amino acidconservation, particularly in the region encompassed by amino acids30-300 (i.e. the N-termini of both proteins).

Second, consensus mapping of homologous functional sites based onestablished ClfA adhesin determinants converged on a specifictopological motif in Als1p. This topological motif is shown in FIG. 26as a cleft formed by the inflection of adjacent facets of two β-sheetdomains.

Third, consistent with primary structure homology, the predictedfunctional cleft motif in Als1p maps to specific residues originatingfrom hypervariable regions in the N-terminal region encompassing aminoacid residues 30-300.

These results provided a structural basis for congruent biologicalfunctions, as well as immunological responses to Als1p and ClfA. Theseresults also further corroborate our overall model of Als1pstructure-activity, and further facilitate targeted approaches tomutational analyses and epitope mapping. Finally, these results indicatethat Als1p and ClfA are adhesins of analogous structure and functionpresent on diverse microbial pathogens.

A monoclonal antibody against S. aureus also was identified that mayreduce infections caused by C. albicans. As with the above structuralfindings, this characteristic also was used to design and implement aneffective vaccine against S. aureus using Als adhesins.

Briefly, a humanized anti-staphylococcal monoclonal antibody (Aurexis®)that is known to recognize surface adhesins on S. aureus is currently inclinical trials. This monoclonal antibody also cross reacts with Alsfamily members. Favorable results of a phase II clinical trial ofAurexis® for the treatment of staphylocbccal bloodstream infections havebeen reported (Inhibitex Inc., 2005; accessed Sep. 19, 2005, athttp://phx.corporate-ir.net/phoenix.zhtml?c=176944&p=irol-newsArticle&ID=707322&highlight=).Briefly, in this report, patients with known S. aureus in the blood wereadministered the Aurexis® antibody as treatment for active infection(i.e., this is not an active vaccine strategy or a prophylaxis study).Nine patients receiving placebo experienced breakthrough bloodstreaminfections caused by Candida, while only three patients in the Aurexis®arm experienced Candida bloodstream infections. Recognizing the decreasein Candida blood infection for those patients treated with an antibodyto S. aureus combined with the above homology and structural findingsindicate that immunogenic epitopes are shared between Candida and S.aureus and that these immunogenic epitopes can be targeted fortherapeutic benefit using immune responses, antibodies or effectormechanisms raised against one species for treatment of the otherspecies. Therefore, the above data together provide for immune responsesto surface adhesins on S. aureus to cross react with Candida spp.

Following the above strategy, exemplary Als adhesin vaccines weredesigned and shown to improve survival of mice infected with S. aureus.The exemplary Als adhesins used to vaccinate were rAls1p-N or rAls3p-N,which were produced and used as described above. Briefly, to determineif these Als vaccines against Candida, rAls1p-N and rAls3p-N, canmediate cross-species protection against S. aureus, female Balb/c micewere vaccinated with the previously described regimen (Complete Freund'sAdjuvant+20 μg of rAls1p-N or rAls3p-N on day 0, followed by a boosterdose in Incomplete Freund's Adjuvant at 3 weeks, both administeredsubcutaneously). Two weeks following vaccination, mice were infected viathe tail-vein with a lethal dose of S. aureus strain 67-0, which ismethicillin-resistant and known to be virulent in animal models. Theresults showing mice survival are shown in FIG. 26. As indicated, boththe rAls1p-N and rAls3p-N vaccines mediated improved long-term survivalin these infected mice (FIG. 27). Additionally, the mechanism ofprotection likely to be an enhancement of Th1 rather than Th2 since nocorrelation between Ab titers and survival of mice vaccinated witheither rAls1p-N or rAls3p-N was observed (FIG. 28).

Example X The Anti-Candida rAls1p-N Vaccine Mediates a Broad Range ofProtection Against Disseminated Candidiasis

This Example show that the rAls1p-N vaccine protects outbred mice fromdisseminated candidiasis, and protects Balb/c mice against othervirulent strains of C. albicans and non-albicans Candida.

The current studies were performed to illustrate the breadth ofprotection induced by rAls1p-N by specifically evaluating its efficacyin outbred mice, in combination with a second adjuvant other thanFreund's adjuvant, against other strains of C. albicans, and againstnon-albicans species of Candida.

Vaccination with rAls1p-N protected outbred mice from disseminatedcandidiasis. Briefly, outbred CD1 mice were obtained from the NationalCancer Institute (Bethesda, Md.). All procedures involving mice wereapproved by the institutional animal use and care committee, followingthe National Institutes of Health guidelines for animal housing andcare. The mice were vaccinated with rAls1p-N+Freund's adjuvant aspreviously described above and in, for example, Ibrahim et al., Infect.Immun. 73:999-1005 (2005); Spellberg et al., Infect. Immun. 73:6191-93(2005). rAls1p-N (amino acids 17 to 432 of Als1p) was produced in S.cerevisiae and purified by gel filtration and Ni-NTA matrix affinitypurification. A high degree of purity (≈90%) was confirmed bySDS-polyacrylamide gel electrophoresis as well as circular dichroism andFTIR, as described above and in, for example, Sheppard et al., J BiolChem 279:30480-89 (2004). Mice were immunized by SQ injection ofrAls1p-N (20 μg) mixed with Complete Freund's Adjuvant (CFA;Sigma-Aldrich, St. Louis, Mo.) at day 0, followed by a booster dose inIncomplete Freund's Adjuvant (IFA; Sigma-Aldrich) at day 21. Controlmice were immunized with CFA/IFA alone. Fourteen days following theboost, immunized mice were infected via the tail-vein with C. albicansSC5314, as we have described previously Ibrahim et al., (2005) supra;and Spellberg et al. (2005), supra. Similar to our previous findings inBalb/c mice, the rAls1p-N vaccine markedly improved the survival ofinfected CD1 mice (FIG. 29A).

Because Freund's adjuvant is considered to be too toxic for use inhumans, we performed a dose response of rAls1p-N vaccine in alum (2%Alhydrogel, Brenntag Biosector, Frederikssund, Denmark), the onlyvaccine adjuvant currently approved by the US Food and DrugAdministration (FDA) for use in humans. Vaccination with alum wasperformed on an identical schedule as Freund's adjuvant, withimmunization on day 1, boost on day 21, and infection 2 weeks later. Wefound that higher doses of rAls1p-N combined with alum resulted insignificant improvements in survival of mice with disseminatedcandidiasis (FIG. 29B). There are also appeared to be a dose responserelationship, with trends to improved survival at higher doses ofrAls1p-N when combined with alum.

The rAls1p-N vaccine also was shown to improve the survival of Balb/cmice infected with several strains of C. albicans. Particularly usefulvaccines utilize an immunogen that can prime the immune system torecognize multiple strains of the target pathogen. By DNA sequenceanalysis, we found that the predicted amino acid sequence of theN-terminal region of Als1p was 99.9% conserved amongst a diverse groupof clinical C. albicans isolates from bloodstream (5 strains), urine (5strains) and oropharyngeal (10 strains) infections (data not shown).These results indicated that the rAls1p-N vaccine can be effectiveagainst a broad array of C. albicans strains. To confirm the breadth ofprotection of the rAls1p-N vaccine against other strains of C. albicans,mice were vaccinated with rAls1p-N+Freund's adjuvant as above, andinfected with one of several clinical isolates of C. albicans (Ibrahimet al., Infect Immun 63:1993-98 (1995)). As shown in FIG. 30, therAls1p-N vaccine significantly improved the survival of mice infectedwith each of these strains.

The rAls1p-N vaccine also was shown to reduce tissue fungal burden inmice infected with several non-albicans species of Candida. Briefly, theALS gene family is present in other Candida species, including C.dubliniensis and C. tropicalis (Hoyer et al., Genetics 157:1555-67(2001)). Similarly, an adhesin analogous to Als family members has beendescribed in C. glabrata (Cormack et al., Science 285:578-82 (1999);Frieman et al., Mol Microbiol 46:479-92 (2002)). To confirm the efficacyof the rAls1p-N against non-albicans species, Balb/c mice werevaccinated with rAls1p-N+Freund's adjuvant as above, and infected viathe tail-vein with C. glabrata 31028 (a clinical bloodstream isolatefrom the microbiology laboratory at Harbor-UCLA Medical Center), C.krusei 91-1159, (generously provided by Michael Rinaldi, San Antonio,Tex.), C. parapsilosis 22019 (clinical bloodstream isolate fromHarbor-UCLA Medical Center), or C. tropicalis. 4243 (clinicalbloodstream isolate from Harbor-UCLA Medical Center). As shown in FIG.31, the rAls1p-N vaccine reduced the kidney fungal burden of miceinfected with each of these species.

In summary, the rAls1p-N vaccine is able to prevent and/or reduce theseverity of an increasingly common and highly lethal disseminatedcandidiasis. The vaccine is efficacious in both inbred and outbred mice,when mixed with alum as an adjuvant, against multiple strains of C.albicans, and against several non-albicans species of Candida. Theseresults further corroborate that the ALS vaccines of the invention areeffective against a wide variety of candidal and other infections.

Example XI The Anti-Candida rAls3p-N Vaccine is Equally Effective asrAls1p-N Against Disseminated and More Efficacious Against MucosalCandidiasis

This Example compares the efficacy of rAls3p-N to rAls1p-N vaccines inmurine models of hematogenously disseminated, oropharyngeal, and vaginalcandidiasis.

Of the ALS family members, the ALS1 and ALS3 genes encode adhesins withthe broadest array of substrate affinity. When compared to one another,Als1p mediated greater adherence to endothelial cells and gelatin, butinferior adherence to epithelial cells (Sheppard et al., J Biol Chem279:30480-89 (2004)). Their differences in adherence qualities suggestedthat rAls3p-N may have different efficacy as a vaccine immunogencompared to rAls1p-N.

The vaccines and vaccinations were performed as described above.Briefly, rAls1p-N and rAls3p-N (amino acids 17 to 432 of Als1p or Als3p)were produced in S. cerevisiae and purified by gel filtration and Ni-NTAmatrix affinity purification, as described above and in Ibrahim et al.,(2005), supra; Spellberg et al., (2005), supra). The amount of proteinwas quantified by modified Lowry assay. A high degree of purity (≈90%)was confirmed by SDS-polyacrylamide gel electrophoresis as well ascircular dichroism and FTIR, as described above and in Ibrahim et al.,(2005), supra; Spellberg et al., (2005), supra). Mice were immunized bysubcutaneous (SQ) injection of 20 μg of rAls1p-N or rAls3p-N mixed withComplete Freund's adjuvant (CFA, Sigma-Aldrich, St. Louis, Mo.) at day0, boosted with another dose of the antigen with Incomplete Freund'sadjuvant (IFA, Sigma-Aldrich) at day 21, and infected two weeksfollowing the boost.

Statistical analyses were performed as follows. The non-parametric LogRank test was utilized to determine differences in survival times of themice. Antibody titers and footpad swelling were compared by the Steeltest for non-parametric multiple comparisons Rhyne et al., Biometrics23:539-49 (1967), or the Mann Whitney U test for unpaired comparisons,as appropriate. Correlations were calculated with the Spearman Ranktest. To determine if heterogeneity existed in replicate survivalstudies, the Kolmogorov-Smirnov test was utilized. P values<0.05 wereconsidered significant.

Vaccination with rAls3p-N was shown to stimulate a broader array ofantibody responses in comparison with rAls1p-N. In this regard, theresults shown in FIG. 32 show mice vaccinated with CFA+rAls1p-N orrAls3p-N developed antibody titers significantly greater than micereceiving CFA alone. Of note, mice vaccinated with rAls3p-N generatedanti-rAls1p-N antibodies at equivalent titers to mice vaccinated withrAls1p-N (FIG. 32, top). In contrast, mice vaccinated with rAls1p-Ngenerated smaller titers against rAls3p-N than did mice vaccinated withrAls3p-N (FIG. 32, bottom). However, both rAls1p-N and rAls3p-N resultedin similar delayed type hypersensitivity responses in vivo as shown inFIG. 33.

The rAls1p-N and rAls3p-N vaccines also were shown to mediate similarefficacy against disseminated candidiasis. Briefly, to furthercorroborate that the rAls3p-N vaccine was as effective as rAls1p-Nagainst hematogenously disseminated candidiasis, mice were vaccinatedwith CFA, CFA+rAls1p-N, or CFA+rAls3p-N, and subsequently infected viathe tail-vein with C. albicans. The results shown in FIG. 34 demonstratethat both the rAls1p-N and rAls3p-N vaccines resulted in significantimprovement in survival.

Correlation of anti-Alsp antibody titers and delayed typehypersensitivity reactions with survival in vaccinated mice subsequentlyinfected with C. albicans was also determined. Briefly, antibody titerswere determined by ELISA in 96 well plates, as we have describedpreviously and in Ibrahim et al., (2005), supra; Spellberg et al.,(2005), supra. Wells were coated with 100 μl per well of 5 μg/mlrAls1p-N or rAls3p-N in PBS. Mouse sera were incubated for 1 h at roomtemperature following a blocking step with tris buffer saline (TBS)(0.01 M TrisHCl, pH 7.4, 0.15 M NaCl) containing 3% bovine serumalbumin. The wells were washed 3 times with TBS containing 0.05% Tween20, followed by another 3 washes with TBS without Tween. Goat anti-mouseIgG secondary antibody conjugated with horseradish peroxidase(Sigma-Aldrich) was added at a final dilution of 1:5000 and the platewas further incubated for 1 h at room temperature. Wells were washedwith TBS and incubated with substrate containing 0.1 M citrate buffer(pH 5.0), 50 mg/ml of o-phenylenediamine (Sigma), and 10 μl of 30% H₂O₂.The color was allowed to develop for 30 min after which the reaction wasterminated by adding 10% H₂SO₄ and the optical density (OD) wasdetermined at 490 nm in a microtiter plate reader. Negative controlwells received irrelevant antibody, and background absorbance wassubtracted from the test wells to obtain final OD readings. The ELISAtiter was taken as the reciprocal of the last serum dilution that gave apositive OD reading (i.e. >mean OD of negative control samples+(standarddeviation*2)).

Delayed type hypersensitivity reactions were assessed by measuring thefootpad swelling tests. Briefly, mice were immunized with rAls1p-N,rAls3p-N, or CFA alone. Two weeks following the boost, baseline footpadsizes of immunized mice were measured using an electronic digitalcaliper. Fifty μg of rAls1p-N or rAls3p-N in 25 μl of PBS were injectedinto the right footpads, and PBS alone injected into the left footpadsof the immunized mice. Twenty-four hours later the footpads were againmeasured. Antigen-specific footpad swelling was calculated as: (rightfootpad thickness at 24 h−right footpad thickness at baseline)−(leftfootpad thickness at 24 h−left footpad thickness at baseline).

Vaccinated mice were bled for titer determinations and underwent footpadswelling tests two days prior to infection. Vaccinated mice that did notsurvive the infection nevertheless had a broad range of antibody titersas shown in FIG. 35. Many such mice had anti-rAls1p-N and anti-rAls3p-Nantibody titers of ≧1:50,000 (≧4.5 log₁₀). As a result, antibody titersdid not significantly correlate with survival. In contrast, theintensity of footpad swelling reactions did correlate with survival(FIG. 35, ρ=0.6 & p=0.009 by Spearman Rank correlation test).

The rAls3p-N vaccine also demonstrated more efficacy than rAls1p-N intwo models of mucosal candidiasis. Because Als3p mediated superioradhesion to epithelial cells compared to Als1p, this observationindicates that rAls3p-N can exhibit unique efficacy in mucosal models ofinfection. The efficacy of rAls1p-N compared to rAls3p-N assessed in asteroid-treated, oropharyngeal model of infection and in a model ofcandidal vaginitis.

Briefly, vaccine studies in the above murine oropharyngeal candidiasis(OPC) model were performed as previously described and as described inSpellberg et al., (2005), supra; Kamai et al., Antimicrob AgentsChemother 45:3195-57 (2001), and Kamai et al., Infect Immun 70:5256-58(2002). Vaccinated mice were immunocompromised by treatment withcortisone acetate (225 mg/kg SQ on days −1, 1, and 3 relative toinfection). On the day of infection, the mice were anesthetized byintraperitoneal injection with 8 mg xylazine and 110 mg ketamine per kg.Calcium alginate urethral swabs were saturated with C. albicans byplacing them in a suspension of 10⁶ organisms per ml in HBSS at 30° C.The saturated swabs were placed sublingually in the oral cavity of themice for 75 min. After 5 days of infection, the tongue and hypoglossaltissue were excised, weighed, homogenized, and then quantitativelycultured to determine the oral fungal burden.

Effectiveness of the vaccine against murine vaginal candidiasis wasperformed by vaccinating female Balb/c mice were treated with 30 μg ofsubcutaneous estradiol valerate dissolved in peanut oil (both fromSigma-Aldrich) on day −3 relative to infection to induce pseudoestrus.On the day of infection, mice were sedated by ip administration of 100mg/kg of ketamine. Sedated mice were infected intravaginally with 10⁶blastospores of C. albicans in 10 μl of HBSS. On day 3 post-infection,vaginas and approximately one centimeter of each uterine horn weredissected en block, homogenized, and quantitatively cultured.

As shown in FIG. 36, in cortisone-treated mice with oropharyngealcandidiasis, the rAls1p-N vaccine mediated a strong trend towardsreduced tongue fungal burden (p=0.054). The overall magnitude of thebenefit was <0.3 log CFU/gram (FIG. 36). In comparison, the rAls3p-Nvaccine mediated a >0.6 log CFU/gram decrease in tongue fungal burdenthat was statistically significant (p=0.005, FIG. 36). Similarly, in anon-immunocompromised model of candidal vaginitis, the rAls3p-N vaccinemediated a 0.7 log CFU/gram decrease in vaginal fungal burden comparedto CFA alone (p=0.02) as shown in FIG. 37. In comparison, rAls1p-Nmediated no benefit at all in the vaginitis model, and rAls3p-N wassignificantly more effective than rAls1p-N (p=0.01).

The above results indicate that a vaccine based on rAls3p-N, which is85% homologous to rAls1p-N at the amino acid level, was equallyeffective against disseminated candidiasis, but was more effective thanrAls1p-N against mucosal infection. The increased effectiveness ofrAls3p-N was seen in both a steroid-treated model of oropharyngealcandidiasis and an immunocompetent model of candidal vaginitis. Theabove results also show achievement of ≧50% long-term survival in amurine model of candidal septic shock with no adjunctive anti-fungaltherapy is encouraging, and further corroborates the therapeutic benefitall ALS vaccines of the invention.

Antibody titers did not correlate with the protective effect of eithervaccine during disseminated candidiasis, but induction of delayed typehypersensitivity in vivo did correlate with protection. These data alsofurther corroborate the mechanism of vaccine-induced protection wasinduction of Type 1, cell-mediated immunity to the fungus. Both rAls1p-Nand rAls3p-N induced equivalent titers of antibody against rAls1p-N, butthat rAls3p-N induced significantly higher titers of anti-rAls3p-Nantibodies than did rAls1p-N. These data indicated that, despite theirhigh degree of amino acid sequence homology (85%), the humoral immunesystem can distinguish between rAls1p-N and rAls3p-N. The above resultsfurther corroborate that, regardless of differences in Als1p and Als3pepithelial cell adherence characteristics, the rAls1p-N and rAls3p-Nvaccines were equally effective in protecting against hematogenouslydisseminated (i.e. endovascular) candidiasis.

In sum, the anti-candidal rAls3p-N vaccine induced equivalentcell-mediated but broader antibody-based responses than did the rAls1p-Nvaccine. The immunogens resulted in an equivalent degree of protectionagainst hematogenously disseminated candidiasis, but rAls3p-N mediatedgreater protection against both oropharyngeal and vaginal candidiasis.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1-11. (canceled)
 12. A method of vaccinating a mammal againstcandidiasis comprising administering to said mammal a pharmacologicallyeffective amount of a vaccine comprising a polypeptide comprising theN-terminal domain of Candida albicans Als3p, or an immunogenic fragmentthereof, in a pharmaceutically acceptable composition.
 13. The method ofclaim 12, wherein said polypeptide comprises said N-terminal domain ofCandida albicans Als3p.
 14. The method of claim 13, wherein saidpolypeptide consists of said N-terminal domain of Candida albicansAls3p.
 15. The method of claim 12, wherein said polypeptide comprisessaid immunogenic fragment of said N-terminal domain of Candida albicansAls3p.
 16. The method of claim 15, wherein said polypeptide consists ofsaid immunogenic fragment of said N-terminal domain of Candida albicansAls3p.
 17. The method of claim 12, wherein said mammal is a human. 18.The method of claim 12, wherein said vaccine is administeredsubcutaneously.
 19. The method of claim 12, wherein said administrationfurther comprises administering a booster dose.
 20. The method of claim12, wherein said composition comprises an immunostimulating adjuvant.21. The method of claim 20, wherein said immunostimulating adjuvant isalum.
 22. The method of claim 12, wherein said candidiasis is mucosalcandidiasis.
 23. The method of claim 12, wherein said candidiasis isdisseminated candidiasis.
 24. The method of claim 23, wherein saiddisseminated candidiasis is hematogenously disseminated candidiasis. 25.The method of claim 12, wherein said candidiasis is caused by Candidaalbicans, Candida parapsilosis, Candida guilliermondii, Candida krusei,Candida dubliniensis, or Candida tropicalis.
 26. The method of claim 12,wherein said polypeptide is produced in Saccharomyces cerevisiae.
 27. Avaccine consisting of a first component and a second component, whereinsaid first component consists of an agglutinin like sequence 3 protein(Als3p), or an immunogenic fragment thereof, and said second componentcomprises an adjuvant in a pharmaceutically acceptable composition. 28.The vaccine of claim 27, wherein said Als3p is from a Candida strainselected from the group consisting of Candida albicans, Candidaparapsilosis, Candida guilliermondii, Candida krusei, Candidadubliniensis, and Candida tropicalis.
 29. The vaccine of claim 27,wherein said first component consists of the N-terminal domain ofCandida albicans Als3p.
 30. The vaccine of claim 27, wherein said firstcomponent consists of an immunogenic fragment of the N-terminal domainof Candida albicans Als3p.
 31. The vaccine of claim 27, wherein saidadjuvant is an immunostimulating adjuvant.
 32. The vaccine of claim 31,wherein said immunostimulating adjuvant is alum.
 33. The vaccine ofclaim 27, wherein said Als3p or immunogenic fragment thereof is producedin Saccharomyces cerevisiae.